SUNDAY, AUGUST 5, 2007 (8.30–10.10)
Session 1: Hemichannels
Session Chair: Ross Johnson
Keynote Speaker:
Michael Bennett: Connexin Hemichannels: They Are Real and Do Things
Michael Bennett1 and Juan Carlos Saez2
1Albert Einstein College of Medicine and 2Pontificia Universidad de Chile
Non-junctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca2+ and is enhanced by low Ca2+, positive membrane potentials, and dephosphorylation of critical residues. Evidence of hemichannel opening includes uptake of molecules that permeate gap junctions but generally not nonjunctional membrane, uptake (or loss) of these molecules proportional to connexin expression, sensitivity of uptake to gap junction blockers and also trivalent cations (La3+, Gd3+), and single channel conductance about twice that of the cell-cell channel. Lowering intracellular redox potential with dithiothreitol (DTT) increases Cx43 hemichannel opening under otherwise normal conditions; increased opening is determined by dye uptake and single channel recording at positive potentials. Both activity measures show block by hemichannel blockers. Biotinylation and Westerns indicate little change in surface expression. In contrast, permeability is increased by metabolic inhibition and surface expression is increased enough to account for the change. DTT and other reducing agents decrease dye uptake under these conditions, rapidly enough to suggest direct action. Cysteines in the cytoplasmic C-terminal domain are potential sites of DTT modification and may mediate these opposite responses. Bath application of reduced glutathione, a membrane impermeant reducing agent, does not increase dye uptake, but glutathione in the recording pipette increases hemichannel opening at positive voltages, suggesting an intracellular site of action. Truncation of the C-terminal that removes the cysteines blocks DTT's effects on dye uptake, but point mutations are necessary to demonstrate mediation by these cysteines. In summary, intracellular redox potential can regulate opening of Cx43 hemichannels, possibly by action on cytoplasmic cysteine residues in its C-terminal. Alterations in redox potential known to occur in physiological conditions may alter hemichannel opening with consequences for paracrine signaling and intercellular interactions.
Connexin Hemichannels, ATP Release and Kidney Function
Janos Peti-Peterdi, Arnold Sipos, and Fiona McCulloch
University of Southern California, United States of America
ATP and metabolites are present in the renal tubular and interstitial fluid and are involved in the autocrine/paracrine purinergic regulation of salt and water reabsorption. Several connexin (Cx) isoforms form large-conductance ATP-permeable hemichannels. We recently localized Cx30 in the apical membrane of renal epithelial cells suggesting it may function as a luminal ATP channel. The purpose of these studies was to test if Cx30 hemichannels release ATP and are involved in the regulation of renal tubular electrolyte and water transport. Wild type and Cx30 knockout mouse cortical collecting ducts were dissected, partially slit-open and microperfused in vitro. Intercalated (IC) and principal cells (PC) were identified based on their selective binding of peanut-lectin (IC, red) or quinacrine (PC, green). A single biosensor PC12 cell, which has purinergic receptors was loaded with the calcium fluorophore Fluo-4, and positioned in direct contact with the apical surface of either IC or PC. Biosensor cell calcium responses, triggered by an increase in tubular flow, were about 4-fold larger when positioned next to IC vs. PC, and were completely abolished with the purinergic receptor blocker suramin (100 μ M). Importantly, biosensor cell responses were completely absent in Cx30−/− mice. Next, mice were surgically instrumented for clearance and pressure natriuresis measurements. Mean arterial blood pressure (MAP) was increased by aorta ligation, and plasma and urine samples were collected for Na, K measurements (flame photometry). In response to step increases in MAP, urine flow increased 4.2-fold in Cx30+/+ compared to only a 2.6-fold increase in Cx30−/− animals. Also, fractional urinary Na+ excretion increased 5.1-fold in Cx30+/+ compared to only a 2.8-fold increase in Cx30−/− animals. This is the first report suggesting that distal tubular Cx30 hemichannels release ATP which inhibits renal salt and water reabsorption causing pressurenatriuresis.
Functional Differences Between Human Cx37 Polymorphic Hemichannels
Jean-Paul Derouette1, Cindy Wong1, Thomas Desplantez2, Isabelle Roth1, Brenda R. Kwak1, and Robert Weingart2
1Geneva University Hospitals, Cardiology and 2University of Bern, Physiology
A genetic polymorphism in the human Cx37 gene (C1019T) has been proposed as potential prognostic marker for atherosclerosis. We have previously reported that Cx37 hemichannels (HC) control the initiation of atherosclerotic plaque development by regulating ATP-dependent monocyte adhesion in apolipoprotein E-deficient mice, an animal model for the disease. The polymorphism in hCx37 results in a non-conservative amino acid change (serine or proline at codon 319) in Cx37. However, the functional significance of this change remains undetermined. In this study, we transfected hCx37-P319, hCx37-S319 or empty pIRES-eGFP vector cDNA into communication-deficient HeLa cells. Control HeLa cells showed increased adherence onto tissue-culture plates as compared to hCx37-expressing cells. Using clones with similar levels of Cx37 expression, we found that P319 cells were less adhesive than S319 cells (18.7 ± 4.6% and 62.9 ± 10.7%, N = 8, P < 0.002). Similar results were obtained in a medium with low calcium concentration. Moreover, reduction in adhesiveness of Cx37-expressing cells was canceled by extracellular apyrase, indicating that the effects are likely mediated by ATP. Conceivably, these effects may reflect differences in HC properties. Therefore, we have measured the electrical properties of Cx37 HC by patch clamp. In Cx37-expressing cells, exposure to low-Ca2+ solution induced a voltage-sensitive current relaxing with time. The analysis resulted in a bell-shaped function gss = f(Vm) (gss: normalized conductance at steady state; Vm: membrane potential) with a peak around −35 mV, suggesting a dual voltage control. Interestingly, steady-state macroscopic conductance (gss) was 1.5-fold increased in P319 cells compared with S319 cells (N = 8). In cell pairs, we observed a decreased main unitary conductance for S319 (∼ 205 pS) compared with P319 (∼ 300 pS) gap junction channels. We hypothesize that the differences in biophysical properties between polymorphic HC may be responsible for inequality in ATP release between S319 and P319 cells, which results in differential cell adhesion.
Pannexin1 Is Part of the Pore Forming Unit of the P2x7 Receptor Death Complex
Gerhard Dahl1, Silviu Locovei1, Eliana Scemes2, Feng Qiu1, and David Spray1
1University of Miami and 2Albert Einstein College of Medicine
The purinergic receptor P2X7 is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X7 receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X7R in Xenopus oocytes results exclusively in formation of a nonselective cation channel. However, here we show that co-expression of P2X7R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X7R co-expressing cells. In co-expressing oocytes extracellular ATP induced dye uptake, which was not observed in oocytes expressing either P2X7 or pannexin1 alone. ATP-induced dye uptake was furthermore suppressed in astrocytoma cells by pannexin1 siRNA. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X7R signaling complex.
SUNDAY, AUGUST 5, 2007 (10.30–13.10)
Session 2: Hemichannels
Session Chair: David Spray
In Vivo Analysis of Undocked Gap Junction Hemichannels in Ovarian Granulosa Cells
Dan Tong, Tony Y. Li, Kathryn E. Naus, Donglin Bai, and Gerald M. Kidder
University of Western Ontario
Targeted knockout of connexin43 in the mouse resulted in impaired ovarian follicle development, suggesting important roles of Cx43 in oogenesis. Cx43 connexons are highly abundant in the membranes of ovarian granulosa cells where they dock to form gap junctions. However, in addition to forming intercellular gap junction channels, gap junction hemichannels can also act as a diffusion pathway for the release of extracellular messengers like ATP. In this study, the presence of undocked Cx43 hemichannels in granulosa cells was demonstrated by fluorescent dye uptake induced either by reduction of extracellular divalent cations or by mechanical stimulation (both conditions are known to cause opening of gap junction hemichannels). ATP release was also detected, and could be abolished by connexin channel blockers. However, neither of these phenomena was observed in Cx43-deficient granulosa cells, indicating Cx43 hemichannels are involved in ATP release and, possibly, in the following intercellular calcium wave. To test the hypothesis that the requirement for Cx43 during folliculogenesis can be fulfilled by hemichannels alone, a Cx43 construct lacking the conserved cysteines on the extracellular loops, reported to form functional gap junction hemichannels but not intercellular channels, was stably delivered into Cx43-deficient granulosa cells by retrovirus. The granulosa cells were then combined with oocytes to make reaggregated ovaries which were grafted into host kidney capsules so that folliculogenesis could be evaluated after four weeks of development in vivo. We found that cysteine-less Cx43 was not able to rescue folliculogenesis in Cx43-deficient ovaries although wildtype Cx43 could, indicating that the formation of intercellular gap junction channels is an essential role of Cx43 in granulosa cells. Thus undocked Cx43 hemichannels in ovarian granulosa cells are involved in ATP release, but they can not alone support follicular development. Funded by the Canadian Institutes of Health Research.
Regulation of Connexin 43-Hemichannels and the Release of Prostaglandins by Mechanical Stress and α5 Integrin
Jean Jiang, Arlene J. Siller-Jackson, Sirisha Burra, Sumin Gu, Stephen E. Harris, and Eugene Sprague
University of Texas Health Science Center at San Antonio
Mechanosensing osteocytes express Cx43 that forms hemichannels on the cell surface. Our previous studies have shown in MLO-Y4 osteocytes that fluid flow shear stress (FFSS) induces the opening of hemichannels through which prostaglandin E2 (PGE2) is released. Here we show that Cx43-hemichannels are closely regulated by mechanical stimulation. FFSS causes the opening of hemichannels and release of PGE2, and both of which were magnitude-dependent with increasing response to increased shear stress. During post-stress periods hemichannels were gradually closed over time, returning to the level of control after 24 h and PGE2 release follows a similar pattern. Hemichannels appear to become increasingly “desensitized,” leading to gradual closure starting after 2 h of continuous shear. However, allowing a rest period between mechanical loadings enhanced the hemichannel response. To identify the potential mediators responsible for the opening of hemichannels, gene microarray analysis was performed showing FFSS up-regulation of α5 integrin that was partially inhibited by the gap junction and hemichannel inhibitor. Immunostaining showed co-localization of α 5 integrin with Cx43 not only on the cell surface but also throughout the cell body and cell processes, becoming more prominent when exposed to FFSS. Co-immunoprecipitation analysis shows the association of α5 integrin with Cx43. Cx43 and α5 integrins were not co-localized with vinculin and paxillin, components of focal adhesions, suggesting that the association between Cx43 and α5 integrin is unrelated to the function of integrins in focal adhesions. More importantly, α5 integrin antibody significantly decreased FFSS induced dye uptake. Together, these results suggest that α5 integrin is a likely mechanical sensor or tether that conveys the effects of mechanical stress on Cx43, leading to the assembly and opening of hemichannels. Opening of hemichannels provides a novel portal for release of prostaglandins and potentially other signaling factors in response to mechanical stress.
Hemichannel Mediated Inositol (1,4,5)-trisphosphates (IP3) Release in the Cochlea
Hong-Bo Zhao and David G. Gossman
Department of Surgery, Otolaryngology, University of Kentucky Medical Center, Lexington, KY
Connexin mutations can induce serious hearing loss. It has been reported that some Cx26 mutations may impair the permeability to inositol (1,4,5)-trisphosphate (IP3) in the cochlea to induce deafness. Here, we report the first direct measurement of hemichannel-mediated IP3 release in the cochlear sensory epithelium by use of fluorescence polarization assay. We found that the reduction of extracellular Ca2+ concentration can induce IP3 release. The IP3 release increased about 2∼ 3-fold as extracellular Ca2+ concentration reduced from 2 mM to 0 mM, and could be blocked by gap junction channel blockers, carbenoxolone, 18-AGA, and octonal. These blockers also reduced the IP3 level at normal extracellular solution (containing 2 mM Ca2+). In addition, the release could be eliminated by pre-incubation of BAPTA-AM to clamp intracellular calcium concentration. The IP3 release was also increased by increase in membrane tension. The response could be blocked by carbenoxolone. The data confirm that connexin channels in the cochlea are permeable to IP3. The data also imply that hemichannels can release IP3 to participate in broad, long-distance intercellular signaling via extracellular pathway in the cochlea. Connexin mutations may interrupt this crucial intercellular signaling function in the cochlea to induce hearing loss. Supported by NIDCD DC05989.
Modulation of Membrane Channel Currents by Gap Junction Protein Mimetic Peptides: Size Matters
Gerhard Dahl, Junjie Wang, Meiyun Ma, Silviu Locovei, and Robert W Keane
University of Miami
Connexin mimetic peptides are widely used to assess the contribution of nonjunctional connexin channels in several processes including ATP release. These peptides are derived from various connexin sequences and have been shown to attenuate processes downstream of the putative channel activity. Yet so far no documentation of effects of peptides on connexin channels has been presented. We tested several connexin and pannexin mimetic peptides and observed attenuation of channel currents that is not compatible with sequence specific actions of the peptides. Connexin mimetic peptides inhibited pannexin channel currents but not the currents of the channel formed by connexins from which the sequence was derived. Pannexin mimetic peptides did inhibit pannexin channel currents but also the channels formed by connexin 46. The channel inhibition by peptides shares commonalities with channel effects of polyethylene glycol, suggesting a steric block as mechanism. These data suggest that reevaluation of data interpretation involving the mimetic peptides is warranted.
SUNDAY, AUGUST 5, 2007 (13.30–15.10)
Session 3: Pannexins and Innexins
Session Chair: Gerhard Dahl
Pannexin 1 Channels Contain a Glycosylation Site that Targets the Oligomer to the Plasma Membrane
Daniela Boassa1, Cinzia Ambrosi1, Gerhard Dahl2, Guido Gaietta3, and Gina Sosinsky4
1University of California San Diego; 2University of Miami School of Medicine; 3University of California San Diego, CRBS; and 4University of California San Diego, Department of Neuroscience
Pannexins are newly discovered channel proteins expressed in many different tissues and most abundantly in the vertebrate central nervous system. Based on membrane topology folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that pannexin1 forms hexamers, reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at N254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two hexamers could dock to form an intercellular channel. Taken together, these findings suggest that Pannexin1 channels do not form gap junctions but rather are distributed throughout the plasma membrane as hexamers. We have started to extend our labeling studies to tissues and have begun acquire large-scale brain maps highlighting the pannexins distribution.
Pannexin1 and Pannexin3 Are Glycoproteins that Exhibit Many Distinct Characteristics From the Connexin Family of Gap Junction Proteins
Silvia Penuela1, Ruchi Bhalla1, Xiang-Qun Gong2, Donglin Bai2, Qing Shao1, and Dale W. Laird1
1Department of Anatomy and Cell Biology, University of Western Ontario and 2Department of Physiology and Pharmacology, University of Western Ontario
Pannexins are mammalian orthologs of the invertebrate gap junction proteins innexins and thus have been proposed to play a role in gap junctional communication. In this study, we cloned and expressed untagged and GFP-tagged mouse Panx1 and Panx3 as well as generated site-directed antibodies to both pannexins to assess whether they exhibited characteristics of the well studied connexin family. Localization of Panx1 and Panx3 in normal rat kidney and N2A cells revealed an even cell surface distribution profile with no evidence of clustering characteristic of connexin-based gap junctions. Western blots of untagged Panx1 revealed multiple species of ∼41–48 kD, while Panx3-expressing cells showed a prominent protein of ∼43 kD. Interestingly N-glycosidase treatment reduced both Panx1 and Panx3 to a single protein band indicating that both pannexins are heavily glycosylated, a property that is distinct from connexins. Moreover, Panx1 and Panx3 are predicted to have a longer half life than Cx43 based on their inability to be cleared from the cell surface upon brefeldin A blockage of protein secretion. Panx1 was found to be expressed in several murine tissues, while Panx3 expression was more limited to skin and cartilage. Finally, Lucifer yellow and intercellular electrical coupling assays strongly suggest that Panx3 is incapable of forming functional homomeric gap junction channels. Collectively, these studies show that Panx1 and Panx3 have many unique characteristics from connexins and Panx3 likely plays a biological role that is distinct from connexinbased gap junctional intercellular communication. Supported by the Canadian Institutes of Health Research and Canada Research Chair Program toDWL and DB. First two authors are co-presenters.
SUNDAY, AUGUST 5, 2007 (15.30–17.10)
Session 4: Life Cycle of Connexins
Session Chair: Dale Laird
Regulation of Cx43 Via Phosphorylation of Specific Residues Is Critical For Epidermal Wound Healing, Myocyte Hypoxia Response and Normal Development
Paul Lampe, Joell L. Solan, and Clarence A. Dunn
Fred Hutchinson Cancer Research Center
We have created several Cx43 phosphospecific antibodies that indicate changes in Cx43 phosphorylation status in tissue during development and wound healing. We found that keratinocytes initially require gap junctional communication (GJC) to initiate cell migration and proliferation to repair wounds but rapidly reduce communication via MAPK phosphorylation at S279/282 that peaks within 1h followed by c-Src dependent phosphorylation at Y247/265 and PKC-dependent S368 phosphorylation peaking at 24 h. Since each phosphorylation event has different effects on GJC, they likely play different temporal roles in regulating wound healing. For example, phosphorylation at S368 causes formation of a specific epidermal basal cell communication compartment with enhanced permeation of larger molecules to apparently regulate keratinocyte migration. We can show that this phosphorylation event also causes a demarcation of neural and pigmented retinal cells in the ciliary margin where stem cell fate determination occurs. PKC has also been shown to be protective in preventing heart damage possibly via dephosphorylation/phosphorylation of Cx43. During ischemia, specific localization of Cx43 to the intercalated disk is lost in a manner coupled to dephosphorylation at several serines but increases on S368. We believe dephosphorylation at S365 to be critical for this process since phosphorylation at S365 and S368 are mutually exclusive. Phosphorylation at S365 and S325/328/330, respectively, are necessary to formthe P1 and P2 SDS-PAGE isoforms of Cx43. Using these antibodies and site-directed mutantswe can showthat the C-terminal 25 amino acids change conformation upon phosphorylation at S365 (via NMR structural studies with Paul Sorgen) and the conformation at prolines 375 and 377 are critical for gap junction formation and ZO-1 interaction. Tissue staining with these antibodies and collaborations with Cecilia Lo using S368A knock-in mice clearly show that temporally regulated Cx43 phosphorylation is critical for proper development and wound repair.
A Novel Cx43 Interacting Protein Involved in Cx43 Proteasomal Degradation
Vivian Su1, Xinli Li1, Wendy Kurata1, Chengshi Jin2, and Alan Lau1
1Cancer Research Center of Hawaii and 2Deparment of Epidemiology and Biostatistics, University of California, San Francisco
Control of connexin43 (Cx43) protein levels can have an important role in regulating cell communication. Cx43 is known to be rapidly degraded with a half-life of 1.5-5 hours. While it has been shown that Cx43 degradation occurs via the lysosomal and proteasomal pathways, how this happens and what controls the degradation is not clear. We have identified a novel Cx43-interacting protein that we have named CIP75. Sequence analysis found that CIP75 contains an ubiquitin-like domain (UbL) and an ubiquitin-associated domain (UBA). Other members of the UbL-UBA family have been demonstrated to interact with proteasomal subunits and thus implicated in proteasomal degradation. We investigated the possibility of CIP75 acting in the Cx43 proteasomal degradation process. Immunofluoresence studies found that CIP75 localizes to the endoplasmic reticulum (ER). In addition, GST pull-down and co-immunoprecipitation experiments demonstrate that CIP75 interacts with components of the proteasome. Further overexpression and knock-down studies suggest that CIP75 is involved in Cx43 turnover in a proteasomal dependent manner. We propose that CIP75 has a role in the ER-associated degradation of Cx43.
Zonula Occuldens-1 Integrates N-Cadherin-Based Cell Adhesion and Connexin43 Gap Junction Dynamics
Andrew Hunter and Robert Gourdie
Medical University of South Carolina
Cadherin-mediated cell adhesion is thought to be required for gap junction formation. Conversely, evidence suggests that connexins participate in the maintenance of cadherin adhesion. Yet the molecular mechanisms that govern interplay between cadherins and connexins at sites of cell-cell contact are largely undefined. ZO-1, an actin-binding MAGUK protein, localizes to both gap junctions and cadherin-based junctions, and thus is a good candidate to mediate interactions between cadherins and connexins at junctional interfaces. We are testing this idea by imaging live HeLa cells transiently co-expressing Cx43-CFP with either N-cadherin-YFP or YFP-ZO-1, in a background of stably expressed untagged Cx43. N-cadherin-YFP colocalized with Cx43 mainly at the periphery of gap junctions, and manifested as dynamic punctae that moved predominately along plaque edges, although some punctae appeared to traverse plaques from edge to edge. Movement of edge-localized punctae often coincided with finger-like protrusions of plaque that appeared to be under tension, as if linked to cytoskeletal dynamics. Consistent with this, cytochalasin treatment depressed the mobility of N-cadherin-YFP punctae and forced plaques into a static, rounded morphology. YFP-ZO-1 also colocalized with Cx43 predominately at plaque edges, although YFP-ZO-1 was distributed more diffusely along edges than N-cadherin-YFP. Interestingly, in the absence of PDZ-mediated interaction with Cx43, ZO-1 continued to target to plaque edges but assumed a punctate distribution similar to N-cadherin; moreover, the protrusive activity at plaque edges was altered. Extensive colocalization of N-cadherin, ZO-1 and actin filaments at the periphery of Cx43 plaques was confirmed by confocal microscopy in fixed cells. These observations support the hypothesis that N-cadherin adhesions target ZO-1 to Cx43 plaques at specialized interfaces that—depending on the specific molecular interactions established by ZO-1—differentially regulate junctional dynamics, in part by modulating linkages to the actin cytoskeleton.
MONDAY, AUGUST 6, 2007 (8.30–10.10)
Session 5: Connexins in the Cardiovascular System
Session Chair: Niels-Henrik Holstein-Rathlou
This session is kindly sponsored by the Danish National Research Foundation Centre for Cardiac Arrhythmia
Cx40 is Crucial for Conduction of Dilations and Renin Secretion and Can Only Be Partially Replaced by Cx45
Cor de Wit1, Stephanie Wölfle1, Volker Schmidt1, Sebastien Alcolea2, Daniel Gros2, Charlotte Wagner3, and Frank Schweda3
1Institute of Physiology, Univ Lübeck; 2Univ. de la Mediterranee, Marseille, France; and 3Physiologisches Institut der Universität Regensburg
In the vasculature intercellular communication via gap junction channels (GJC) is reflected by the conduction of vasomotor responses. GJC in arterioles are composed of Cx40, 37, 45 and 43. We investigated whether Cx40 can be functionally replaced by Cx45 using mice in which Cx40 is replaced by Cx45 (Cx40KI45) that has a smaller conductance. Propagation of dilations and constrictions initiated by acetylcholine (ACh), bradykinin (Bk), or KCl were studied in arterioles (∼33 μm) of the cremaster muscle by intravital microscopy in Cx40-deficient (Cx40ko), Cx40KI45, and wildtype (wt) mice. Arterial pressure and plasma renin concentration were measured in intact animals, renin secretion in isolated perfused kidneys. ACh induced a similar dilation in all genotypes at the stimulation site (58–69%) which was conducted to a 1200 μm distant site with an amplitude of 54 ± 8% in wt. The remote dilation was, however, reduced to 29 ± 4% in Cx40ko and similarly impaired in Cx40KI45 (21 ± 4%). Likewise, conducted dilations initiated by Bk were reduced in Cx40ko and Cx40KI45. In contrast, the propagation of KCl-induced constrictions was comparable in all genotypes. Arterial pressure was strongly elevated in Cx40ko (168 ± 2 vs. 118 ± 3 mm Hg in wt), but only moderately in Cx40KI45 mice (130 ± 8 mm Hg). Plasma renin was elevated 5.1-fold in Cx40ko compared to wt. Renin secretion was suppressed at high perfusion pressure in wt kidneys but not in Cx40ko. Nevertheless, the difference in arterial pressure between Cx40ko and wt persisted after ACEinhibition (130 ± 3 vs. 108 ± 2 mm Hg). Cx40 function is critical for the conduction of dilations and supports pressure feedback on renin secretion in the kidney. Endothelial function of Cx40 cannot be replaced by Cx45 suggesting that specific Cx-properties are necessary in this context. However, other functions (related to blood pressure control) may be partially restored by Cx45.
Loss of Connexin40 Alters Renin Production, Causing Hypertension
Jacques-Antoine Haefliger1, Krattinger Nathalie2, Capponi Alessandro3, Lucia Mazzolai2, Pascal Nicod2, and Paolo Meda4
1Department of Medicine, Lausanne University Hospital, CHUV; 2Department of Medicine, University Hospital, Lausanne; 3Department of Medicine, University Hospital, Geneva; and 4Department of Cell Physiology and Metabolism, University of Geneva, School of Medicine, CMU
The renin-producing cells, which play a major role in the control of blood pressure, are coupled to each other and to endothelial cells by Connexin40 (Cx40) channels. We report that Cx40 deficient mice (Cx40−/−), but not their heterozygous littermates, are hypertensive due to increased numbers of renin-secreting cells (RSC), renin biosynthesis, and plasma renin. Treatment with inhibitors of the renin-angiotensin system reduced the blood pressure of Cx40−/− mice to values observed in wild type controls. The elevated blood pressure of Cx40−/− mice was not affected by clipping one renal artery (2K1C, renin-dependent model of hypertension) or after a high salt diet. Under these conditions, however, Cx40−/− mice showed altered production and release of renin. The renin mRNA ratio between the clipped and the contralateral kidney of 2K1C Cx40−/− mice was lower than in 2K1C WT controls, indicating that the response to a change in local blood pressure was altered. Furthermore, the blood pressure of Cx40−/− mice was significantly increased in the 1K1C renin-independent model of hypertension, suggesting a synergistic effect of the renin- and volume-dependent mechanisms. The loss of Cx40 was not compensated in RSC by either Cx43 or Cx37. Moreover, Cx37 was not detected in the renal endothelial cells of Cx40−/− mice and was reduced in RSC, indicating that coexpression of Cx37 and Cx40 is important for clustering of gap junction channels in these cells. The data show that renin secretion is dependent on Cx40, and stress the value of Cx40−/− mice as a genetic model of renindependent hypertension.
Hypertension Differentially Regulates Connexin Expression in the Mouse Aorta
Florian Alonso1, N. Krattinger1, A. Simon2, A. Capponi3, L. Mazzolai1, P. Nicod1, P. Meda4, and J.-A. Haefliger1
1Department of Medicine, Lausanne University Hospital, CHUV; 2Department of Physiology, College of Medicine, University of Arizona; 3Department of Internal Medicine, HUG, University of Geneva; and 4Department of Cell Physiology and Metabolism, CMU, University of Geneva
Gap junctions play a role in the contractility of the aorta wall. Here, we investigated the effects of chronic hypertension on connexin expression in the aorta of transgenic mice lacking Cx40 (Cx40-/-), which represent a genetic model of hypertension due to increased renin secretion. After 4 weeks of renin-dependent hypertension, as induced in the 2-kidney, 1 clip model [2K,1C], Cx40−/− mice exhibited the same elevated mean blood pressure (MBP), as operated wild type mice and non operated Cx40−/− mice. Conversely, 4 weeks after induction of a renin-independent hypertension (1-kidney, 1 clip model [1K,1C]), or administration of NG-nitro-L-arginine methyl ester [L-NAME] that inhibits nitric oxide synthase, MBP was significantly increased in Cx40−/− mice compared to untreated controls, suggesting a synergistic effect of the renin-dependent (2K1C) and -independent (1K1C, L-NAME) mechanisms. All hypertensive mice featured a similar increase in the thickness of the aortic wall, and expressed Cx43 in the smooth muscle cells (SMC) of the media layer. However, when compared to controls, renin-dependent (Cx40−/− and 2K1C WT), but not renin-independent (1K1C and L-NAME) hypertensive mice featured an upregulation of aortic Cx43. In contrast, all hypertensive mice featured increased expression of Cx37, Cx40 and Cx45. Strikingly, Cx37 immunostaining was decreased in endothelial cells of the aorta of hypertensive animals, suggesting that this connexin was induced in the hypertrophied SMC. Blockade of the renin-angiotensin system with candesartan (an AT1-receptor antagonist) or ramipril (an angiotensin converting enzyme inhibitor), normalized the blood pressure and the thickness of the aortic wall of Cx40−/− mice, while decreasing connexin expression in this vessel. The data show that connexins are differentially regulated by the mechanical forces induced by high blood pressure, and participate in the molecular adaptation of the aortic wall which is observed under these pathological conditions.
MONDAY, AUGUST 6, 2007 (10.30–12.10)
Session 6: Connexins in the Cardiovascular System
Session Chair: Nick Severs
This session is kindly sponsored by the Danish Cardiovascular Research Academy
Prominent Role for Connexin37 in Haemostasis and Thrombosis
Brenda Kwak1, Pierre Fontana2, Isabelle Roth1, Rocco Sugamele3, Marc Chanson4, and Anne Angelillo-Scherrer3
1Geneva University Hospitals, Cardiology; 2Geneva University Hospitals, Angiology and Hemostasis; 3Lausanne University Hospitals, Hematology; and 4Geneva University Hospitals, Pediatrics
Formation of platelet plug initiates haemostasis at sites of vascular injury and triggers pathological thrombosis in ischemic tissue disease. However, the mechanisms of inter- and intracellular signalling leading to the formation of stable platelet plugs are still poorly understood. Here, we report the expression of Cx37 in mouse and human platelets. We also observed neurobiotin diffusion between platelets isolated from human or wild-type mice during platelet aggregation in vitro. In contrast, the tracer did not spread within the platelet aggregates obtained from Cx37-deficient mice. Significant differences in the cessation of bleeding in vivo were obtained after tail transsection in Cx37−/− and Cx37+/+ mice: bleeding time was 3.8 ± 0.5 min for Cx37−/− and 8.8 ± 1.4 min for Cx37+/+ mice (mean ± SEM, n = 20, P < 0.005). In addition, in vitro platelet aggregation in response to collagen and ADP was increased in Cx37−/− compared to Cx37+/+ mice and in Cx37+/+ platelets pre-treated with 200 microM Cx37 blocking peptides (n = 4, P < 0.05). In agreement, pre-incubation with 50 microM 18-alpha glycyrrhetinic acid (aGA) increased (P < 0.05) the aggregation of human washed platelets with each agonist used: median maximal aggregation without or with aGA were 0% and 84% for arachidonic acid, 42% and 67% for ADP and 17% and 59% for collagen. We also observed remarkable differences in the time of survival after inducing thrombo-embolism by injecting a collagen/epinephrine mixture into the jugular vein: control mice survived 4.2 ± 0.4 min and Cx37−/− mice only 1.9 ± 0.3 min (n = 8, P < 0.001). Finally, we recorded thrombus formation by intravital microscopy in the mesenteric arteries after vessel wall injury by FeCl3. We found that rhodamine-6G-labeled platelet recruitment at the site of the vascular injury was faster in Cx37−/− than in Cx37+/+ mice (n = 4). Taken together, our results demonstrate that Cx37 plays a crucial role in the physiological and pathological signalling in platelets leading to haemostasis and thrombosis.
Cell-Cell Coupling Between Stem Cells and Adult Cardiac Myocytes: From Intercellular Flow of Pacemaker Current to Action Potential Generation
Virgis Valiunas1, G. Kanaporis1, L. Valiuniene1, C. Gordon1, J. Zuckerman1, R.B. Robinson2, M.R. Rosen2, I.S. Cohen1, and P.R. Brink1
1Department of Physiology and Biophysics, Stony Brook University and 2Department of Pharmacology, Columbia University
Human mesenchymal stem cells (hMSCs) over expressing a pacemaker gene mHCN2 introduced into canine ventricular myocardium induce pacing as a result of formation of functional gap junctions with host tissue cells. This suggests hMSCs may be attractive as a cellular delivery system to any syncytia. The aim of this study was to determine what constitutes sufficient cell-cell coupling of pacemaker current (If) intercellular flow from hMSC to adult myocyte to generate an action potential (AP). To test this notion, acutely isolated adult canine ventricle myocytes were co-cultured with hMSCs or HeLaCx43 cells transfected with mHCN2 gene. Cell-cell coupling etween heterologous pairs of cardiac myocytes and hMSCs or HeLaCx43 cells was evaluated using double whole cell patch clamp. Electrical coupling increased with time in co-culture and the average junctional conductance was ∼ 1nS/ ∼ 2nS after 24 hours, ∼ 6 nS/∼ 5 nS after 36 h and ∼ 14 nS/ ∼ 12 after 54 h for myocyte-hMSC/myocyte-HeLaCx43 cell pairs, respectively. When junctional coupling between an mHCN2 transfected cell and myocyte cell exceeded gj of ∼ 4nS, hyperpolarization of the myocyte cell exhibited If –currents, indicating current transfer from mHCN2 expressing cell to myocyte via gap junctions. The magnitude of the If currents recorded in myocytes increased with increasing junctional conductance. With junctional conductances > 5 nS, activation of sufficient If current in the mHCN2 transfected cell generated an AP in the neighboring coupled ventricle cell. Hence, we demonstrate that pacemaker currents delivered to the cardiac myocyte via gap junctions from a genetically engineered (mHCN2-expressing) cell contribute to generation of the action potential. Further the critical electrical cell-cell coupling between cardiac and mHCN2 transfected cells is exceeded within 24 to 36 hours, allowing the cell pair to function as a pacemaker unit. Supported by AHA 0335236N, NIH GM5526, HL28958, Boston Scientific.
MONDAY, AUGUST 6, 2007 (13.30–15.10)
Session 7: Connexins in the Cardiovascular System
Session Chair: Eric Beyer
This session is kindly sponsored by the Danish Heart Foundation
Regulation of Cx43 in Myocardial Infarction: A Role for Hypoxia Inducible Factor 1
Heather S. Duffy1, Juan Carlos Osorio2, Edward J. Ciaccio2, and James Coromilas2
1Columbia University, College of Physicians and Surgeons and 2Columbia University
Gene array analysis of culturedmyocytes has indicated that chronic hypoxia (11% O2) downregulates Connexin 43 (Cx43). We hypothesized that hypoxia induced HIF-1 (a transcriptional regulator) regulates Cx43 following myocardial infarction (MI). To test our hypothesis we used neonatal rat ventricular myocytes treated with dimethyloxaloglycine (DMOG), a specific HIF-1 agonist. Using Western Blot analysis we determined that DMOG treatment (10 mM) induced increases in HIF-1α and concurrent decreases in Cx43. Additionally, immunofluorescence studies show DMOG treatment caused HIF-1α nuclear translocation and Cx43 remodeling. Thus increased HIF-1α leads to remodeling of Cx43 in cultured myocytes. To test in vivo effects of HIF-1α on Cx43, we infused DMOG into normal ventricle through the Left Anterior Descendent Artery (LAD). Infusion ofDMOGincreased epicardial HIF-1α and caused Cx43 remodeling in normoxic tissue comparable to that found in the epicardial border zone (EBZ) myocytes after MI. To determine if HIF-1α is associated with Cx43 loss following myocardial infarction (MI), we examined HIF-1α and Cx43 levels at 3hr, 48 hr and 5 days post-infarction. Western blot of post-CO epicardial border zone (EBZ) showed increased HIF-1α as early as 3 hr and persisting to 5 days. This was associated with a decrease and lateralization of Cx43. To determine if HIF-1α associated changes in Cx43 translate into functional changes in the intact heart, we did conduction mapping of the epicardial surface of normal hearts infused with DMOG which indicated that HIF-1α associated decreases in Cx43 correlated with slowing of conduction, similar to changes seen in early MI. These data indicate that HIF-1 activation leads to Cx43 remodeling and plays a role in altered cardiac function following MI. This suggests that transcriptional regulation of Cx43 by HIF-1 plays a role in pathophysiological remodeling of cardiac Cx43 and underlies changes seen in conduction properties following MI.
Down-Regulation of Connexin43 Gap Junctions in Human Congestive Heart Failure Is Associated with an Increased Interaction of Connexin43 with ZO-1
Alexandra Bruce, Stephen Rothery, Emmanuel Dupont, and Nicholas J Severs National Heart and Lung Institute, Imperial College London
Remodeling of gap junction organization and connexin43 (Cx43) expression contributes to the pro arrhythmic substrate in human heart disease, but little is known of the mechanisms underlying this remodeling. One candidate regulatory factor is the PDZ MAGUK protein, zonula occludens-1 (ZO-1), which interacts with the carboxy terminus of Cx43. Left ventricular myocardium from undiseased control hearts (5 samples) was compared with that of explanted hearts from transplant patients with end-stage heart failure due to idiopathic dilated cardiomyopathy (DCM) or ischaemic cardiomyopathy (ICM; 5 samples from each group). Using immunoconfocal and immunoelectron microscopy, we determined that ZO-1 is specifically localized at the intercalated disk. No ZO-1 signal is detected at the lateral plasma membrane of the myocyte and there is no alteration of distribution in diseased tissue. When compared to controls, there is a significant increase in ZO-1 protein levels in both DCM (P > 0.0001) and ICM (P = 0.039) groups; however, mRNA levels in the failing ventricle (results pooled as no significant difference detectable between groups) are not significantly different (P = 0.18). Double immunolabeling demonstrated that while substantial areas of the intercalated disk are labeled only for ZO-1 or only for Cx43, a proportion of the ZO-1 label is co-localized with Cx43 and is therefore situated for potential interaction. Quantitative analysis of immunolabeling in en-face intercalated disks demonstrates that the co-localization of Cx43 with ZO-1 is significantly increased in the failing left ventricle (pooled results; P < 0.0001). Co-immunoprecipitation experiments confirmed interaction between Cx43 and ZO-1. The amount of Cx43 that co-immunoprecipitates with ZO-1 (as a proportion of total Cx43) is significantly increased in the failing heart in both the Triton X-100 soluble (P = 0.01) and Triton X-100 insoluble (P = 0.01) fractions. Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.
MONDAY, AUGUST 6, 2007 (15.30–17.10)
Session 8: Connexins in Development, Reproduction and Organogenesis
Session Chair: Elke Winterhager
This Session is kindly sponsored by the Danish Cancer Society
Reversal of the Transformed Phenotype of Hela Cells is Specifically Mediated by Cx26 Gap Junction Intercellular Channels Through Cell Cycle Block
Bruce Nicholson1, Anjana Chandrasekhar1, and Shoshanna N. Zucker2
1University of Texas Health Science Center at San Antonio and 2State University of New York at Buffalo
Exogenous connexin (Cx) expression has been shown to suppress growth and tumorigenesis in several transformed cell lines, yet no comprehensive picture of how connexins regulate cell growth has emerged. Using the HeLa model system, we have confirmed previous findings that, while three different connexins (Cx43, Cx32, and Cx26) are each efficiently expressed, and mediate robust dye transfer between cells, only Cx26 clones consistently showed reduced growth in low serum and anchorage independent conditions. However, unlike previous studies, we have shown that this effect requires the presence of functional Cx26 intercellular channels. No growth suppression is observed when Cx26T135A (a mutant which forms normal gap junction structures, but lacks any channel activity), nor Cx26R75Y (a mutant that readily forms hemichannels but not gap junction channels), are expressed at comparable levels to wt Cx26. This indicates that there are unique characteristics to the permeability of Cx26 channels that allow the transfer of signals between cells that mediate suppression of growth in low serum. This suppression results from partial blocks in both G1 and G2 cell-cycle phases, as assessed through flow cytometry at different times after addition of serum to serum-starved cultures. A comparison of several signaling pathways that could potentially regulate these steps showed that Cx26 cell coupling induces sustained up-regulation of PKA, JNK and ERK pathways. A common link in the regulation of each of these pathways is cAMP, which has recently been shown to pass more efficiently through Cx26 gap junction channels than those of Cx32 and 43 (see Toloue and Nicholson abstract at this meeting). Thus, this system may provide the first comprehensive picture of how intercellular communication through gap junctions regulates transformed growth, and underscores the importance of including the role of gap junctions in the analysis of signaling networks.
Decreased Levels of Cx43 Results in a Delayed Ductal Development and in Impaired Milk Expulsion in Mammary Glands of Gja1jrt/+ Mice
Isabelle Plante, Crystal S. Lounsbury, and Dale W. Laird
University of Western Ontario
Branching morphogenesis during the development of the mammary gland is fundamental for the process of milk synthesis and expulsion. In mice, hormones influence ductal branching during puberty but the role of gap junctions in mammary gland morphogenesis is still poorly understood. Expression of Cx26, Cx32 and Cx30 has been demonstrated in luminal epithelial cells of the mammary gland while Cx43 is expressed in myoepithelial cells. This project aims to determine the role of Cx43 in the normal development of the mammary gland by employing a mouse model of oculodentodigital dysplasia (Gja1Jrt/+), that harbors an autosomal dominant Cx43 mutation, and its wild-type (WT) littermates. Even though female Gja1Jrt/+ mice are fertile, litter's survival is low, suggesting impaired gland development, milk production or expulsion. Mice were killed at critical stages of mammary gland development (4, 6, 10 weeks old) and at parturition. Cx43 protein levels were greatly reduced in the mammary glands of Gja1Jrt/+ while no changes in Cx43 mRNA levels were observed. Mammary glands were significantly smaller and a delayed development of the ductal network was observed in Gja1Jrt/+ mice before and during puberty (4 and 6 weeks old). At adult age (10 weeks), while the glands were still smaller, one population of Gja1Jrt/+ mice exhibited poor ductal development while a second population was not significantly different from WT. At parturition, morphology of the glands didn't differ between Gja1Jrt/+ and WT. Interestingly, milk was present in the mammary glands of mutant mice and Western blot analysis showed an increase in beta-casein levels suggesting that Gja1Jrt/+ mice properly produce milk, but it was not expelled. Together, these data showed that decreased levels of Cx43 results in a delayed development of the mammary gland and defects in the expulsion of milk. Supported by the CIHR, CBCRA and a CIHR-STP to IP.
Connexin43 Is Required for Normal Skeletal Development and Bone Mass Acquisition
Roberto Civitelli1, Marcus Watkins1, Bertrand Guillotin1, David Ornitz1, Radoslaw Dobrowolski2, and Klaus Willecke2
1Washington University, St. Louis and 2Universität Bonn
Connexin43 (Cx43) is the major gap junction protein expressed in osteoblasts and osteocytes. Because genetic deletion of the Cx43 gene (Gja1) results in perinatal death, we have used a Gja1flox/LacZ gene replacement model and Credriving promoters to ablate Gja1 at different stages of osteoblast differentiation. Using the α 1(I) collagen promoter, we have reported reduced peak bone mass, osteopenia, and osteoblast defects, whereas deletion using osteocalcin promoters only minimally affects bone mass. In this study, we used the Dermo-1 (DM1) promoter, which is expressed in chondroosteoprogenitors at E11.5. DM1-Cre;Gja1–/flox mice are viable and of normal size at birth, but with age they remain substantially smaller than their littermates (body weight: −38% at 1 month, −18% at 6 months). Radio-absorptiometric analysis revealed significantly lower than normal bone mineral density in DM1-Cre;Gja1–/flox mice (−15% at 2 months), persisting up to 6 months. On histology, X-galpositive cells were observed in DM1-Cre; Gja1–/flox mouse tibias up to 6 months, selectively localized along the endocortical and trabecular surfaces, the primary spongiosa, but excluding cells in the bone marrow space. Trabecular bone mass and cortical thickness were both reduced, −57% and −37% respectively, in conditionally deleted animals relative to wild type littermates. Growth plate width was also significantly decreased (−34%), although proliferative and hypertrophic chondrocytes were represented. These animals also exhibited significantly smaller and narrower skulls, remindful of craniofacial malformations reported in germline Gja1 null mice and in the Gja1 linked human disease oculodentodigital dysplasia. Thus, embryonic Gja1 ablation in osteo-chondroprogenitor cells results in craniofacial malformations, stunted skeletal development and severe post natal osteopenia. Skeletal abnormalities and post-natal growth defects are more severe than those occurringwhen Gja1 is deleted later in osteoblast differentiation, revealing a prominent role of Cx43 at early steps of osteogenesis.
TUESDAY, AUGUST 7, 2007 (8.30–10.15)
Session 9: Connexins in Brain and Nervous Tissue
Session Chair: David Paul
Removal of the C-Terminus of Cx43 Results in Enhanced Damage During Stroke
John Bechberger1, G.R. Bechberger2, M. Suen2, M. Kozoriz2, K. Maass3, K. Willecke4, and C.C. Naus2
1Lab Supervisor, University of British Columbia; 2Department of Cellular and Physiological Sciences, the University of British Columbia, Vancouver, BC, Canada; 3Department of Pharmacology, SUNY Upstate Medical, 4Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Bonn, Germany
The function of astrocytic gap junctions and their connexin proteins in ischemic or metabolic insults remains controversial. We have previously shown that blocking gap junctions enhanced excitotoxic neuronal injury in vitro. We have also shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion (MCAO) exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. Furthermore, mice in which the human GFAP promoter-driven cre transgene deletes the floxed Cx43 coding DNA in astrocytes also exhibit enhanced injury after MCAO. These results suggest that astrocytic Cx43 is important for the regulation of neuronal apoptosis and gliosis following stroke, and support the view that astrocytes play a critical role in neuroprotection during ischemic insults. Recently, it has become apparent that Cx43 exhibits not only cell to cell communication and hemi-channel activity, but also protein—protein interaction at its C-ternimus. To further define this protein cytoplasmic binding role of Cx43, we have carried out MCAO experiments in mice expressing a Cx43K258stop, where a translational stop codon has been inserted at position 258 of the Cx43 amino acid sequence (Mass et al., 2004). These mice exhibit a further enhancement of the cerebral injury following stroke, implicating the need for the C-terminal in mediating neuroprotection. Astrocyte cultures from these mice reveal reduced dye coupling and Ca wave propagation. Therefore the presence of C-terminus of Cx43 is important during ischemic conditions. This work was funded by the Canadian Institutes of Health Research (CIHR) and Heart and Stroke Foundation of Canada to C.C.G.N., and by a grant of the German Research Association through the Research Group on Keratinocytes to K.W.
A Role for Connexin43 in the Spread of Damage after Spinal Cord Injury
Simon J. O'Carroll, Colin R. Green, and Louise F. B. Nicholson
University of Auckland
Within 24–48 hours after severe spinal cord injury (SCI), inflammation and damage spread occurs, leading to a significant increase in the size of the area affected. This spread is propagated by a gap-junction-mediated bystander effect, whereby gap junction channels spread neurotoxins and calcium waves from the site of damage to otherwise healthy surrounding tissue. SCI also causes opening of uncoupled connexin hemichannels leading to an exacerbation of the damage. We have previously demonstrated, using rat models of spinal cord injury that antisense oligodeoxynucleotides to connexin43 (Cx43) prevent swelling and cell death. In this study we investigated whether mimetic peptides designed against the extracellular region of Cx43 could reduce swelling and cell death after SCI. A number of mimetic peptides were designed against the extracellular region of Cx43. Peptides were incubated with excised rat spinal cord segments in an ex vivo culture system. The amount of swelling was determined macroscopically; in cultures that showed reduced swelling the numbers of neurons and astrocytes were analysed using immunohistochemistry. One of the peptides significantly reduced the degree of swelling when compared with treatment using a control peptide or media alone. Immunohistochemical analysis using markers for neurons (SMI-32) and astrocytes (GFAP) demonstrated that application of the peptide resulted in an increased survival of neurons and a significant reduction in astrogliosis compared with control cultures. Use of a mimetic peptide against Cx43 prevents swelling, neuronal cell death and astrogliosis following SCI. We believe this effect is mediated by inhibiting the opening of Cx43 hemichannels and/or by preventing the gap-junction-mediated bystander effect after spinal cord injury.
TUESDAY, AUGUST 7, 2007 (10.30–12.10)
Session 10: Connexins in Brain and Nervous Tissue
Session Chair: Christian Naus
The Role of Gap Junctions in Neuronal Migration and Cortical Development
Cima Cina, John F. Bechberger, and Christian C.G. Naus
University of British Columbia
During embryonic development, young neurons migrate from the ventricular zone (VZ) to the cortical plate (CP) of the cerebral cortex. Disturbances in this neuronal migration have been associated with numerous diseases such as mental retardation. One possible cause of these neuropathologies is an aberration in normal gap junctional communication. We have previously shown that Cx26, Cx36, Cx37, Cx43, and Cx45 are expressed in developing mouse neocortex. Of these, Cx26 and Cx43 are expressed by radial glial progenitor cells in the VZ which give rise to neurons and provide scaffolding for their migration. To assess the implication of Cx26 and Cx43 in neuronal migration, we used GFAP-Cre and nestin-Cre mice which have been crossed with either Cx26fl/fl or Cx43fl/fl mice. Our immunolabelling suggest that the removal of Cx43 or Cx26 from radial glial progenitors in conditional knockout mice at embryonic day (E) 18 alters the normal lamination of intermediate zone and the CP. We introduced a Tα 1-promoter driving GFP construct into the VZ of E14 conditional knockout mice using electroporation. The electroporated embryos were allowed to develop to day 18 in utero and subsequently brains were examined for cell migration defects. In conditional knockout mice, the transfected cells had migrated from the VZ and accumulated in the intermediate zone, whereas in normal mice, neurons had migrated to the CP suggesting that deletion of Cx43 in progenitor cells causes a delay in neuronal migration. Subsequently, we were able to rescue neuronal migration in conditional knockout mice by inserting GFAP promoter driving Cx43 constructs into the lateral ventricle of mouse neocortex at the onset of neuronal migration (E14). This study will provide an understanding of tissue specific null mutation of Cx genes and the ensuing effect on neuronal migration in the brain. This research is supported by Canadian Institutes of Health Research.
Zebrafish Cx55.5(C54X) “Knock Outs” Have Deficits in Cone-Mediated Vision
Maarten Kamermans, Trijntje Sjoerdsma, Jan Klooster, Yvonne Claassen, and Colleen Shields
Netherlands Institute for Neuroscience, Retinal Signal Processing Group, Amsterdam, The Netherlands
Cx55.5 hemichannels in the tips of horizontal cell dendrites may function in horizontal cell negative feedback to cone photoreceptors in the zebrafish retina. In order to test for a link between Cx55.5 hemichannels and the feedback mechanism, we obtained a zebrafish with a cytosine to adenine point mutation at nucleotide position 162 in the Cx55.5 coding sequence. This mutation creates a premature stop codon where a cysteine residue would normally occur. These fish are effectively Cx55.5 “knock outs”, as only the first of the four transmembrane domains are possibly translated. Aside from the absence of Cx55.5 protein, there are no morphological differences in the retinal network of homozygous mutants compared to wild type clutch-mates. Measurement of optokinetic responses for low spatial frequencies shows significantly reduced sensitivity in homozygous mutants relative to wild types. This deficit in sensitivity seems to result from the absence of Cx hemichannel-mediated feedback to cones. This study is the first to demonstrate a change in the visually-guided behaviour of a living, behaving animal by altering the retinal horizontal cell network.
WEDNESDAY, AUGUST 8, 2007 (8.30–10.10)
Session 11: Channel Structure and Function
Session Chair: Bruce Nicholson
Structural Analysis of Four Connexin26 Mutant Gap Junctions and Hemichannels
Gina Sosinsky1, Cinzia Ambrosi2, Joshua Brown2, Amy Smock2, Michael Schmid3, and Atsunori Oshima4
1NCMIR, Department of Neurosciences, UCSD; 2University of California, San Diego; and 3Baylor College of Medicine; and 4Kyoto University
Connexin26 (Cx26) is the second smallest of the gap junction proteins and one of the most ubiquitous members. It is highly expressed in many major organs where Cx26 gap junctions play critical roles in homeostasis. We are particularly interested in four single site mutations found in the transmembrane helices (M1-M4) that cause pore abnormalities: (1) Cx26T135A: a closed pore mutant with a single amino acid change in M3; (2) Cx26M34A: a decreased activity mutant, originally identified in non-syndromic hereditary deafness with a site specific mutation in M1; (3) Cx26P87L: a rectifying channel with a site specific mutation in M2; and (4) Cx26V84L: a pore mutant in a residue in M2 identified in non-syndromic deafness that eliminates the passage of a signaling molecule, IP3, while retaining dye coupling and voltage gating capabilities. We use a baculovirus-Sf9 expression system to express and purify Cx26 hemichannels for biochemical and electron microscopic analysis. These constructs have a His6 tag preceded by a thrombin cleavage site for protein purification using Ni-NTA beads. We are also applying methods of His-tagged purification to obtain gap junctions and have found an increase in purity combining a detergent-based and a His-tag purification protocol. All four mutants make “normal” appearing gap junctions that are indistinguishable from wild-type, however, upon dodecylmaltoside solubilization, Cx26T135A and Cx26P87L hexamers are unstable. P87L hexamer formation and stability can be rescued by double infecting Sf9 cells with both wild-type and P87L baculoviruses. These results suggest that the T135 and P87 residues may be in regions of subunit-subunit contact while M34A and V84L are not. To study the physical pore size and shape of these mutant channels, three-dimensional structures are generated by electron tomography of individual membranes combined with volume averaging methods. Supported by NIH grants GM065937 (GES), RR04050 and NSF grant MCB-0131425 (GES).
Three Dimensional Structure of a Human Connexin26 Gap Junction Channel Reveals a Plug in the Vestibule
Atsunori Oshima1, Kazutoshi Tani1, Yoko Hiroaki1, Gina E. Sosinsky2, and Yoshinori Fujiyoshi1
1Kyoto University and 2University of California, San Diego
Connexins (Cx) are the proteins that form the membrane bound cell-cell communication channels, gap junctions, whereby electronic coupling and permeation of the small molecules are essential processes of multicellular lives. Cx26 is the second smallest member of the gap junction family proteins and related to human diseases such as skin disorders and hearing loss. Here, we report the projection and the 3D structures of a mutant human Cx26 (hCx26M34A). We over-expressed a hexahistidine tagged hCx26M34A using baculovirus recombinant expression system. Detergent solubilized and purified hemichannels were reconstituted into the synthetic lipid (DOPC) to produce two-dimensional crystals with the unit cell parameters of a = 112.4Å, b = 111.2Å, gamma = 90.0° and the phase relationships of p22121 symmetry. Four untilted images taken by cryo-electron microscopy were processed and merged at 7Å resolution. The projection map displays hexameric channels each of which shows a weak but significant density in the channel pore. While crystallizations were done with hemichannel preparations, the 3D map at 10Å resolution in membrane plane revealed that two hemichannels had re-docked at their extracellular surfaces into the full intercellular channel and this crystal form contained two sets of symmetry related intercellular channels and three lipid bilayers. The 3D map clearly revealed a prominent new density which resided in each hemichannel pore and had contact with the inner most helices of surrounding subunits at the bottom of the vestibule, strongly suggesting that the channel was closed physically and directly by a pore plug. The density map demonstrates that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.
Residues Participating in Pore-Lining and Gating Converge
Vytas Verselis1, M. Pilar Trelles2, Qingxiu Tang3, Thaddeus A. Bargiello3, and Miduturu Srinivas2
1Albert Einstein College of Medicine; 2Department of Biological Sciences, SUNY State College of Optometry, New York, NY; and 3Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx NY
Sensitivity of gap junction channels to transjunctional voltage independent of the membrane potential implies that gating elements reside in the pore where they can sense the local field. Our previous work using the substituted cystein accessibility method (SCAM) on single Cx46 and Cx50 hemichannels identified a pore-lining segment corresponding to a region predicted to extend from midway within the first transmembrane span (TM1) into the first extracellular loop (E1). Despite high overall sequence homology, we find Cx46 and Cx50 differ substantially in gating characteristics, particularly lacking the strong voltage and Ca2+-dependent gating, provisionally termed loop gating, which effectively closes Cx46 hemichannels upon membrane hyperpolarization. Mutagenesis studies indicate that pore-lining residues near the TM1/E1 border are important in conferring this difference in loop gating. Mutation of individual positions in this region to cysteine in Cx50 produced functional hemichannels, but currents associated with these hemichannels potentiated substantially upon reduction with DTT consistent with disulfide bond formation. Oxidation reversed potentiation, but was hindered when hemichannels were maintained open by membrane depolarization and low extracellular Ca2+. Cys-substiution of pore-lining residues deeper in TM1 did not show sensitivity to DTT. All positions, however, could be modified by methanethiosulfanate (MTS) reagents added from extracellular or intracellular sides. Use of a large MTS reagent, MTSEA-Biotin-X, revealed six sequential reductions in single channel current consistent with accessibility of all six subunits. These data suggest that the pore diameter in the open state of Cx50 hemichannels is wide enough to accommodate six biotin moieties and that gating involves a substantial movement of pore lining residues at TM1/E1 border that results in narrowing of the pore such that alpha-carbon separation can be reduced to ∼5.6 Å.
Measuring the Selectivity and Rate of Natural Metabolite Transfer Through Gap Junctions of Different Composition
Masoud Toloue and Bruce Nicholson
University of Texas Health Science Center San Antonio
Knowledge of actual signals and the rate at which they are transferred between cells by gap junctions has long stood as an impediment to developing a detailed understanding of the function of different connexins in various organs. To bridge this “gap”, we have developed a method for measuring single channel flux rates of natural metabolites through gap junctions composed of different connexin isotypes expressed in Xenopus laevis oocyte pairs. Initially single channel flux rates of a non-hydrolysable variant of ATP (ATPγ S) were measured for Cx26, 32 and 43 channels, and found to vary by over an order of magnitude (104 to 1.3 × 105 molecules/channel/sec, after normalization to a 1 mM concentration gradient), with Cx43 channels being the most permeable. These assays were then expanded to include radiolabelled AMP and cAMP. By reducing the temperature to 8°C, and including the phosphodiesterase inhibitorIBMXin the case of cAMP, metabolic conversion of these permeants could be largely prevented over the course of the experiment. A comparison of the fluxes of these three metabolites through Cx26, 32 and 43 channels, after normalization for concentration gradient and temperature, yielded several novel conclusions. ATP was consistently 2.5 fold less permeable than AMP in all connexins, presumably reflecting the differences in size of these two permeants. However, these metabolites were 5-15 fold more permeable through Cx43 than either Cx26 or Cx32 channels. By contrast, cAMP was 5–6 fold more permeable through Cx26 than Cx43 or 32 channels. These results clearly demonstrate that different connexins form channels that show specific preferences for metabolites. The subtlety of this distinction is most evident in the 30 fold difference in permeability of AMP and cAMP through Cx43 channels, indicating that these channels can distinguish a single phosphodiester bond.
Independent Regulation of Electrical and Chemical Communication Via Cx43 Gap Junctions
Nathanael Heyman and Janis Burt
University of Arizona
Recently published data comparing permeability of Cx43 comprised gap junctions to either positively or negatively charged dyes and electrical conductance (permselectivity) across the same gap junction showed variability that exceeded two orders of magnitude in the relationship of these parameters. To ascertain whether this variable permselectivity is explained by variable charge or size selectivity of the comprising channels, the permeability of individual Cx43 gap junctions to dyes of differing size or charge was determined using dual dye injection techniques. The resulting data indicate that Cx43 junctions are size but not charge selective and do not show significant variability in either parameter. The absence of such variability suggests that the large range of junctional permselectivities previously reported is not due to an equivalently large range of individual channel selectivities. Rather, the data suggest that open Cx43 channels exist in one of two states, dye-permeable and dye-impermeable, both of which remain conductive to smaller ions. Regulation of the proportion of channels in each of these states would not alter junctional dye selectivity but would alter junctional permselectivity, which could represent a mechanism bywhich cells independently control electrical and chemical communication via Cx43 gap junctions. To investigate whether the Cterminus (CT) or phosphorylation of S368 therein influences dye selectivity we measured selectivity of junctions composed of Cx43-S368A or Cx43tr (terminus removed). Although the permselectivity of Cx43-S368A junctions is substantially reduced relative to wildtype, no change in dye selectivity (size or charge) was observed for either Cx43-S368A or Cx43tr. These data suggest that while the CT and phosphorylation of S368 therein may be involved in conversion of channels between dye permeable and impermeable states, neither significantly affects selectivity of dye permeable channels.
WEDNESDAY, AUGUST 8, 2007 (10.30–12.10)
Session 12: Channel Structure and Function
Session Chair: Gina Sosinsky
Mechanism of c-Src Disruption of the Connexin43/Zonula Occludens-1 Interaction
Paul Sorgen, Fabien Kieken, Admir Kellezi, and Bethany J. Hirst-Jensen
University of Nebraska Medical Center
Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of molecular partners. In particular, c-Src can disrupt the connexin43/ZO-1 interaction, leading to down-regulation of intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely distant connexin43 domains; however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we used a biophysical approach to determine the mechanism of c-Src-mediated connexin43/ZO-1 dissociation. Structural studies indicated that the c-Src SH3 domain induced multiple conformational changes to the connexin43 carboxyl terminal domain (Cx43CT), however, the physical interaction alone, does not account for the disruption of the Cx43CT/ZO-1 PDZ-2 domain interaction. Further studies identified a direct SH3/PDZ-2 interaction, the affinity of the SH3/PDZ-2 interaction is higher than that between the Cx43CT/PDZ-2 and Cx43CT/SH3, and many PDZ-2 residues that interact with SH3 are also involved in the Cx43CT interaction. These results suggest that dissociation of the Cx43/ZO-1 complex occurs, in large part, due to a c-Src/ZO-1 interaction, facilitated by the inherent flexible Cx43CT.
Caveolin-1 and -2 Interact with Cx43 and Regulate Gap Junctional Intercellular Communication
St'ephanie Langlois1, Kyle N. Cowan2, Qing Shao1, Bryce J. Cowan4, and Dale W. Laird1,3
Departments of 1Anatomy and Cell Biology, 2Surgery, and 3Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada and Department of 4Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada
It has been reported that connexin 43 (Cx43) interacts with caveolin-1 (Cav-1), but the role of this association and whether other Cavs bind Cx43 has yet to be established. To assess the role of the interaction of Cx43 with Cav-1 and possibly with Cav-2, we used rat epidermal keratinocytes (REK), which express these proteins endogenously. REKs express Cx43 at the plasma membrane and in intracellular compartments thus allowing future investigation of a role of Cavs in constitutive Cx43 trafficking and/or internalization. Using co-immunoprecipitation assays, we demonstrate that the various phosphorylation species of Cx43 (P0, P1, P2) interact with Cav-1 and Cav-2, thus identifying Cav-2 as a new Cx43 binding partner. High resolution confocal microscopy and Brefeldin A treatment suggest that a population of Cx43 co-localizes with Cavs at the plasma membrane and in the Golgi apparatus. Triton X-100 fractionation and sucrose gradient centrifugation indicate that a pool of Cx43 is enriched in lipid rafts with Cav-1 and -2. Treatment with methyl-β-cyclodextrin reduces the Cx43/Cavs association, suggesting that a population of Cx43 interacts with Cavs in lipid rafts/caveolae. Mutation or deletion of the C-terminal tail of Cx43 inhibits its association with Cav-1 and -2, indicating that this domain is required for the Cx43/Cavs interaction. Treatment with methyl-β-cyclodextrin and RNAi knockdown of Cav-1 reduce gap junctional intercellular communication (GJIC) as assessed by dye transfer assays. Over-expression of Cav-1 in 293T cells, which express Cx43 but are deficient in caveolins, enhanced GJIC. Altogether, these data indicate that Cx43 interacts with Cav-1 and -2 in the Golgi and at the plasma membrane in lipid rafts through a mechanism that involves its C-terminal domain. Cav-1 and -2, most likely through their association with Cx43, play a role in regulating GJIC. Supported by the Canadian Institutes of Health Research to DWL and a CIHRSTP to SL.
Functional Characterisation of Connexin Co-expression: Cellular and Molecular Tools
Priyanthi Dias, Neil Thomas, Giselle Rowlinson, Alberto Cabestrero de Diego, Nicholas J. Severs, Kenneth T. Macleod, and Emmanuel Dupont
National Heart and Lung Institute, Imperial College
In mammalian hearts, connexin43 (Cx43), Cx40 and Cx45 are co-expressed in varying amounts in different regions of the myocardium. For instance, the conduction system expresses different connexin combinations with Cx45 predominating in the proximal part (AV node) and high levels of Cx43 and Cx40 in the distal part (Purkinje fibres). The functional correlates of connexin coexpression are difficult to elucidate in vivo owing to the various cell morphologies/physiology in the conduction system, the technical difficulties of measuring conduction velocities in specific regions of the heart and the impossibility of changing levels of expression. To circumvent these problems, we have engineered rat liver epithelial (RLE) cells that endogenously express Cx43 to co-express Cx40 or Cx45 in response to ponasterone A (0.1-2 μ mol/l). At maximal induction, similar amounts of transfected and endogenous connexins are expressed. Dye transfer is significantly reduced but input resistance of the monolayer is maintained. Using Tx100 solubilization assays, amounts of junctional connexons were lower in Cx40 transfectants and higher in Cx45 cells indicating that RLE cells maintain electrical coupling through changing the number of heteromeric, docked connexons. Since conduction velocities cannot be measured in RLE cells, we are using the differentiated atrial cell line HL-1. Through sub-cloning we have obtained clones (#2 and #6) with different conduction velocities (using micro-electrode arrays, #2: 1.62 +/− 0.6mms-1 (SEM), #6: 17.7 +/− 0.1mms-1 (SEM)). These results correlate well with levels of Cx40 and Cx43 expression (#2, total Cx40+43 17 arbitrary units (au), junctional Cx40+43 8.25au, ratio Cx40/43 1.2; #6, total Cx40+43 53au, junctional Cx40+43 32.6au, ratio Cx40/43 2.1). Electroporation with plasmids and subsequent antibiotic selection or siRNA suppression of connexin expression are feasible. The HL-1 clones are therefore an ideal cell model for studying the contribution of active and passive membrane properties and connexin expression to action potential propagation.
WEDNESDAY, AUGUST 8, 2007 (13.30–15.10)
Session 13: Connexins and Pannexins in Skin and Sensory Organs
Session Chair: Jean Jiang
Acceleration of Wound Healing and Inhibition of Mesenchymal Transition after Corneal Endothelial Injury In Vivo by Connexin43 Knockdown
Masahito Oyamada, Ping Dai, Shigeru Kinoshita, Tetsuro Takamatsu, and Yukiko Nakano
Kyoto Prefectural University of Medicine
The corneal endothelium is extremely vulnerable to injury because it lacks a robust proliferative response after injury and undergoes endothelial-mesenchymal transition/transformation (EMT) leading to corneal fibrosis and eventual loss of visual acuity. Appropriate treatment for corneal endothelial injury that circumvents the lack of proliferative response and inhibits EMT, although needed, has yet to be developed. Here, we explore connexin43 (Cx43) knockdown approach as an efficient treatment for corneal endothelial injury using an in vivo rat scrape injury model. Scrape injury to the corneal endothelium was caused using a 30-gauge needle, and immunolabeling for ZO-1, alpha-smooth muscle actin (alpha-SMA), and Cx43 was performed to analyze changes in Cx43 expression during wound healing. A single injection of Cx43 antisense oligodeoxynucleotide (AS-ODN) or small interfering (si)RNA was applied into the anterior chamber simultaneously with injury, and the wound closure was examined by ZO-1-immunolabeling and propidium iodide staining. Cell proliferation was studied by Ki67 immunolabeling. Complete wound closures were observed in 5/6 corneas on day 3 post-injury with either Cx43 AS-ODN or siRNA treatment, whereas no complete closure was observed on day 3 in the controls [sense-ODN (0/6) or nonsense siRNA (0/6)]. Cx43 knockdown increased the Ki67-positive proliferating cell number on day 3 post-injury, whereas it decreased the number of alpha-SMA-positive myofibroblasts on day 5. Furthermore, even without injury, Cx43 knockdown treatment decreased Cx43 expression and induced Ki67-positive proliferating endothelial cells in vivo. These results show that Cx43 knockdown induces corneal endothelium proliferation but inhibits EMT during wound healing in vivo, suggesting that Cx43 knockdown might be a new therapeutic approach not only for acceleration of wound closure but also for prevention of fibrosis in various organs by inhibiting endothelial- or epithelial-mesenchymal transitions.
Pannexin1 Localization in the Horizontal Cell/Photoreceptor Complex of the Zebrafish Retina
Georg Zoidl1, Nora Prochnow1, Jan Klooster2, Stefanie Bunse1, Maarten Kamermans2, and Rolf Dermietzel1
1Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, University Street 150, D-44801 Bochum, Germany and 2Research Group Retinal Signal Processing, Netherlands Brain Research Institute, Amsterdam, The Netherlands
Pannexins (Panx) constitute a novel family of gap junction type proteins. The localization of Panx1 at postsynaptic sites in hippocampal and cortical principal neurons (Zoidl et al., Neuroscience, 2007) and the previously reported electrophysiological characteristics of Panx1 hemichannels suggest that they may function in neurons of the brain as non-junctional channels (pannexons). Here we addressed the role of Panx1 in the retina. Panx1 expression has been detected in horizontal cells and ganglion cells. Since horizontal cells form distinct and highly specialized asymmetric neuronal contacts we used the zebrafish retina as a model to investigate the identity, localization and electrophysiological characteristics of zebrafish Panx1 in the horizontal cell/photoreceptor complex. We cloned the zebrafish pannexin1 expressed in the retina and ruled out the existence of other Panx1 isoforms predicted by public databases. Next we generated an antibody specific for this protein. The mRNA and protein expression analysis of zebrafish pannexin1 confirmed the widespread expression previously observed for murine and human pannexin1. Light and electron-microscopical analysis showed that Panx1 is expressed in horizontal cells and forms hemichannels in dendrites of the horizontal cell/photoreceptor complex in vivo. Transiently transfected Neuro2A cells were used to overexpress Panx1 in vitro and to characterize the hemichannel properties. In summary, this study demonstrates that in the zebrafish a second gap junction type protein with hemichannel properties co-exists aside of Cx55.5 in the horizontal cell/photoreceptor complex. Supported by DFG and SFB 509 “Neurovision” (R.D.; G.Z.), the Graduate College 736 (S.B.) and KNAW (M.K.).
Direct Physical Interaction with Major Intrinsic Protein/Aquaporin-0 Facilitates the Surface Expression of Cx50 in Differentiating Lens Fibers
Jean Jiang and Jialu Liu
University of Texas Health Science Center at San Antonio
Lens is dependent upon a network of gap junctionmediated intercellular communication for the exchange of ions and metabolites to maintain lens transparency and homeostasis. Three connexins are expressed in the vertebrate eye lens, from which two of them are in lens fibers. In our previous studies, we observed that one of chick lens fiber connexins, chCx50 associates with major intrinsic protein (MIP)/aquaporin-0(AQP0) in differentiating lens fibers and this association disappears in mature lens fibers. Moreover, the C-terminus of MIP/AQP0 directly interacts with the intracellular loop (IL) domain of chCx50. However, the functional significance of this interaction in the lens is unknown. Here, we generated recombinant retroviruses containing chCx50, chCx43, and chimera chCx50×46(IL) with the replacement of IL domain of chCx50 with that of chCx46, another lens fiber connexin. Like wild type connexins, chCx50×46(IL) is capable of forming functional gap junctions. These retroviruseswere injected into lens vesicles at early embryonic stages, and the injected lenses were isolated right before hatching. Interestingly, we observed that exogenous chCx50 nonetheless was predominantly expressed on the membranes of lens fiber cells where its endogenous counterpart was localized, even though expression of connexin mRNA was driven by the retroviral promoter. Exogenous chCx43, on the other hand, is primarily localized in epithelium cells. To determine the role of MIP and chCx50 interaction in lens fiber, we injected recombinant retrovirus containing chCx50×46(IL) into lens vesicle. In contrast to cell surface localization of chCx50, majority of this chimera was retained in the cytoplasm of lens fiber cells. These results suggest that MIP is likely to facilitate the targeting and stabilization of chCx50 on the cell surface of differentiating lens fibers. These data suggest a role of MIP/AQP0 in the facilitation of assembly and formation of gap junctions during the early lens development.
THURSDAY, AUGUST 9, 2007 (8.30–10.10)
Session 14: Pharmacology of Gap Junctions
Session Chair: Patricia Martin
This session is kindly sponsored by Wyeth
Keynote Speaker
Stefan Dhein: Pharmacology of Gap Junctions
Stefan Dhein
Clinic for Cardiac Surgery, University of Leipzig
Gap junctions provide the basis of direct intercellular communication and, thus, play a key role in regulating tissue, organ and organism function, as they are involved in the control of growth, differentiation and cell function. Thus, in most diseases, certain aspects are related to gap junction function or malfunction. A number of diseases is directly related to mutations of gap junction genes such as Charcot Marie Tooth disease, others are related to gap junction uncoupling, such as certain cardiac arrhythmias, or –at least in parts-tumor growth of malignoms. On that background, pharmacology seeks for possibilities to modulate gap junctional intercellular communication. The intercellular gap junction conductance g(GJ) depends on the number of channels (N), the single channel conductance yj, and the open probability P according to g(GJ) = N yj P. In consequence, pharmacological tools can either modulate the number of channels via alteration of the expression or the electrical conductance of one or more connexin isoforms. There are a number of agents producing acute uncoupling (e.g. arachidonic acid, heptanol, octanol, palmitoleic acid, certain eicosanoids, Ca++, Na+, H+), while there are others acutely improving coupling (e.g. certain eicosanoids, antiarrhythmic peptides like AAP10, ZP123 (= Rotigaptide), epicatechin, forskolin, eicosapentaenoic acid) via either enhanced phosphorylation of Cx, enhanced incorporation of channels into the membrane or other effects. Another group of agents such as angiotensin (via AT1-receptors), endothelin (via ETA-receptors) noradrenaline, phenylephrine (via α 1D-adrenoceptors), isoprenaline (via β 1/β 2-adrenoceptors) or statins can up-regulate Cx expression, while others such as nicotine down-regulate the number of channels, as well as agents interfering with the docking, assembly or degradation process. This opens perspectives for the development of new drugs, but also gives new aspects for old drugs like beta blockers, ACE-inhibitors or digitalis. Thus, substances enhancing coupling might open new perspectives for antiarrhythmic drug treatment or for limiting metastatic growth. Reference: Salameh & Dhein, BBA 2005, 1719: 36-58
Aminosulfonate Inhibition Requires the Carboxyl-Terminal Domain of Connexin26
Darren Locke1, Liang Tao2, and Andrew L. Harris3
1NJMS-UMDNJ; 2Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong Province, P. R. China; and 3Department of Pharmacology and Physiology. New Jersey Medical School, UMDNJ, Newark, New Jersey
Protonated aminosulfonates directly and reversibly inhibit homomeric and heteromeric Cx26-containing hemichannels, but not homomeric Cx32 hemichannels. Defined structural requirements for the ligand indicated interaction of amine and sulfonate groups with connexin. However, prior studies did not determine which connexin domains were involved, if amine/sulfonate binding sites were on both Cx26 and Cx32 in heteromeric hemichannels, and if junctional channels were aminosulfonate-sensitive. These questions were addressed using channels composed of Cx26 and/or Cx32, with and without cleavable carboxyl-terminal tags. Hemichannel activity was assessed in liposomes, and in cells by dye uptake through unpaired hemichannels opened at low extracellular calcium. In contrast to wild-type hemichannels, heteromeric Cx26Tag/Cx32 and homomeric Cx26Tag hemichannels were not aminosulfonate-sensitive. However, Cx26/Cx32Tag hemichannels were aminosulfonate-sensitive. Carboxylterminal tagging of Cx26 blocked aminosulfonate-sensitivity, as inhibition was restored for tag-cleaved Cx26Tag/Cx32 hemichannels (Cx26Tc/Cx32) and observed for non-tagged homomeric Cx26 hemichannels. Cx26/Cx32Tc hemichannels remained aminosulfonate-sensitive, but, surprisingly, homomeric Cx26Tc hemichannels remained aminosulfonate-insensitive. Instead, aminosulfonate narrowed rather than closed Cx26Tc channels; tag cleavage leaves four amino acids at the carboxyl-terminus. Thus, short carboxyl-terminal addition to Cx26 blocks full aminosulfonate-mediated pore closure, unless hemichannels are heteromeric; the uncleaved tag blocks aminosulfonate-sensitivity in all channel configurations. Aminosulfonate effect on junctional channels was assessed by intercellular dye-coupling. Taurine substantially reduced dye-coupling through non-tagged Cx26 and Cx26/Cx32Tag channels, but not through Cx26Tag, Cx26Tag/Cx32, or Cx32Tag channels. Taurine-induced inhibition of Cx26/Cx32Tag and nontagged Cx26 channels was blocked by extracellular HEPES, a membrane-impermeable aminosulfonate that blocks the taurine membrane transporter. These studies show that aminosulfonate-induced pore closure utilizes the Cx26 carboxyl tail, and that junctional channels have the same aminosulfonate-sensitivity as corresponding hemichannels. Supported by NIH grants GM36044 & GM61406.
Effect of “RXP-E”, a Peptide that Interferes with Gap Junction Closure, on the Electrophysiology of Neonatal Rat Cardiac Myocytes
Rebecca Lewandowski, Junko Shibayama, Wanda Coombs, David Auerbach, Viviana Muñoz, Sergei Mironov, José Jalife, Steven Taffet, and Mario Delmar
SUNY Upstate Medical University
Gap junctions provide a low resistance pathway for electrical propagation. In the heart, slow and meandering conduction will occur upon gap junction uncoupling. This altered conduction can be arrhythmogenic in nature. The role of gap junction regulation on the genesis of arrhythmias is unclear, however, largely due to the limited availability of pharmacological tools to manipulate gap junction function. We have recently shown that a peptide (dubbed “RXP-E”) that binds to the carboxyl terminal domain of Cx43, partly prevents octanol-induced and acidification-induced uncoupling in Cx43-expressing N2a cell pairs (Shibayama et al, Circ Res. 2006). Here, we have characterized the effect of RXP-E on action potential propagation in monolayers of neonatal rat ventricular myocytes. RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) sequence that allowed for its translocation to the intracellular space (Kim et al, Exp Cell Res 2006). The peptide was fluorescein-tagged to allow for visualization by fluorescence microscopy. Thirty minutes after incubation, fluorescein could be detected in nearly 100% of the cells. Synchronization of spontaneous electrical activity within the monolayer was studied by optical mapping. Heptanol superfusion (2 mM) caused the loss of action potential propagation in control preparations. In contrast, synchronous and homogeneous propagation was maintained in monolayers pre-treated with RXP-E. Additional experiments showed that RXP-E also prevents uncoupling in pairs of cardiac myocytes, and that it can pulldown Cx43 out of a heart lysate, thus demonstrating the interaction of RXP-E with the native gap junction protein. Overall, these data indicate that RXP-E: a) associates with cardiac Cx43, b) can be delivered into multicellular cardiac preparations, c) interferes with heptanol-induced and acidification-induced uncoupling and d) can prevent heptanol-induced conduction block. RXP-E may serve as a tool to characterize the importance of conductive gap junctions in the function of multicellular systems, including the heart.
THURSDAY, AUGUST 9, 2007 (10.30–12.10)
Session 15: Pharmacology of Gap Junctions
Session Chair: Stefan Dhein
This session is kindly sponsored by Zealand Pharma
Discovery of Novel Potent and Selective Antiarrhythmic Peptides—From AAP10 To Stable, Orally Available Small Molecules
Bjarne D. Larsen1, Carsten Boye Knudsen1, John Butera2, and Jørgen Søberg Petersen1
1Zealand Pharma and 2Wyeth Research
AAP10 is a potent gap junction modifying molecule of the 1st generation of synthetic antiarrhythmic peptides (AAPs), but due to poor stability, AAP10 was unsuitable as a drug. The strategy to increase the enzymatic stability of the hexapeptide AAP10 was based on substitution of L- for D-amino acids and terminal acetylation and amidation and the use of the back-bone retro analogue to compensate structurally for the inversion of the overall chirality. The 2nd generation molecule rotigaptide is very stable towards enzymatic degradation (half life is 14 days in human plasma) and it is now in clinical development for ventricular arrhythmias. A pharmacophore model was created based on structure-activity-relationship (SAR) studies using a hexapeptide library. NMR studies allowed determination of intermolecular distances between pharmacophores. The objective of this study was to develop a 3rd generation of small molecules with similar activity to AAP10 and rotigaptide that would allow oral administration. Small peptides are absorbed in the gut through the peptide transporter PEPT1 and therefore a library of tripeptides and modified dipeptides were designed to fit both the pharmacophore model for activity and the PEPT1 transporter. This work resulted in several stable, potent and orally available small antiarrhythmic peptides, and among those ZP1210 H-D-Lys(4-Nitrobenzoyl)-Gly-OH, ZP1219 H-D-Asn(NH(4-nitrobenzyl))-Ala-OH, ZP1248 H-D-Asn(NH(4-methoxybenzyl)-Ala-OH, ZP1149 (Hydroxyacetyl-Asn-Tyr-NH2) were identified. AAP10, rotigaptide and the modified small peptides have similar distance between chemical pharmacophores (5.4–6.9 Angstrom) and they all delay calcium chloride-induced cardiac conduction block in mice with similar maximal efficacy. In rats, the oral bioavailability of these molecules is: AAP10 (0%), rotigaptide (2%), ZP1149 (1.2%), ZP1210 (32%), ZP1219 (15%), and ZP1248 (29%). In summary, SAR from AAPs, NMR data and screening of a small peptide library have led to the identification and characterization of new structural classes of orally active small molecule gap junction modifiers possessing remarkable stability and potency.
Pharmacological Characterization of GAP-134, an Orally Bioavailable Small Molecule Gap Junction Modifier, in Mouse, Rat and Canine Arrhythmia Models
James K. Hennan1, Kun Liu1, Gwen Morgan1, Robert Swillo1, John Butera1, Joel Kantrowitz1, Ketil Haugan2, Bjarne Due Larsen2, and Jorgen S. Petersen2
1Wyeth Research and 2Zealand Pharma
GAP-134 is an orally bioavailable gap junction modifier with similar activity as the hexapeptide development compound, rotigaptide. We studied the effect of GAP-134 on stress-induced conduction slowing in rat atrial strips, on cardiac conduction block in mouse atria and on reperfusion arrhythmias and infarct size in dogs. In atrial strips subjected to metabolic stress GAP-134 significantly prevented conduction velocity slowing at 10 nM (5.6 ± 9.4%) and 100 nM (0.4 ± 5.0%) compared to controls (−29.1 ± 5.3%). In mice administered an intravenous infusion of CaCl2 (200mg/kg/min), GAP-134 significantly delayed the time to onset of cardiac conduction block at doses ranging from 10-12 mol/kg to 10-9 mol/kg, with peak efficacy at 10-11mol/kg (206 ± 21% vs. vehicle). After oral dosing a similar delay in time to conduction block was observed (10mg/kg, 207 ± 10%). GAP-134 was studied in dogs subjected to ischemia (60min) and reperfusion (4 hr) injury. GAP-134 was administered 10-min before reperfusion as a bolus (μ g/kg) + IV infusion (μ g/kg/hr) at doses of: 0.25 + 0.19 (n = 6); 2.5 + 1.9 (n = 7); 25 + 19 (n = 6); 75 + 57 (n = 5); and vehicle control (n = 7). Premature ventricular complexes (PVC's) were quantified during reperfusion with four or more consecutive PVC's defined as ventricular tachycardia (VT). Total incidence of VT was reduced significantly with the two highest doses of GAP-134 (1.7 ± 0.8; 2.2 ± 1.4 events; p < 0.05) compared to controls (23.0 ± 6.1). Total PVC's were reduced significantly from 11.1 ± 1.6% in control animals to 2.0 ± 0.7% and 1.8 ± 0.8% after the two highest doses of GAP-134. Infarct size, expressed as percent of left ventricle, was reduced significantly from 19.0 ± 3.5 in controls to 7.9 ± 1.5 and 7.1 ± 0.8% (p < 0.05) at the two highest doses of GAP-134. In conclusion these results demonstrate that GAP-134 is a potent antiarrhythmic compound with cardioprotective effects and potential utility in both atrial and ventricular muscle.
Rotigaptide Alters the Gating Inactivation Properties of Ventricular Gap Junctions
Richard Veenstra and Xianming Lin
SUNY Upstate Medical University
To investigate whether rotigaptide can alter the kinetic gating and steady state inactivation and recovery properties of ventricular gap junctions, we preincubated cultured neonatal murine ventricular cardiomyocytes with clinical pharmacological doses of rotigatide and measured steady state and time-dependent gap junctional currents (Ij) using the dual whole cell voltage clamp technique. Overnight exposure to 35 – 350 nM rotigaptide increased the normalized steady state minimal gap junctional conductance (Gmin) by about 10% with maximal effects occurring at about 100 nM. The gating charge valence (z) or half-inactivation voltage (V1/2) were unaltered. During the 1/sec ventricular action potential voltage clamp experiments, the kinetics of inactivation were slowed and the maximal inactivation during the action potential plateau was reduced from 0.46 of peak Gj under control conditions to 0.54 by 100 nM rotigaptide. In Cx40KO ventricular myocytes, 35 nM rotigaptide reduced the maximal inactivation from 0.40 to 0.62, suggesting that it is most effective at treating Cx43 gap junctions. No effect of rotigaptide treatment on the facilitated recovery of Gj was observed. Kinetic analysis of the fast and slow inactivation components of wild-type ventricular Ij revealed no change in the Vj-dependence of inactivation, but the fast and slow inactivation time constants at 70 mV Vj were increased from 112 and 675 ms to 148 and 1287 ms by 100 nM rotigaptide. These data provide the first direct evidence that rotigaptide treatment mostly slows the rate but also reduces the amount of inactivation that occurs during slow ventricular conduction of the cardiac action potential. Slow conduction and unidirectional block are prerequisites for the establishment of reentrant activity and these experimental results may help explain the beneficial effects of rotigatide on reentrant forms of ventricular tachycardias that often lead to fibrillation and sudden cardiac death.
Abstracts from Poster Presentations (LISTED ALPHABETICALLY BY FIRST AUTHOR)
Program for Poster Presentations
SUNDAY, AUGUST 5, 2007 (19.30–22.00)
Poster Session 1
Session 1.1: Hemichannels
Session 1.2: Pannexins and Innexins
Session 1.3: Life Cycle of Connexins
Session 1.4: Regulation of Channel Activity
MONDAY, AUGUST 6, 2007 (20.00–22.30)
Poster Session 2
Session 2.1: Connexins in the Cardiovascular System
Session 2.2: Connexins in Development, Reproduction and Organogenesis
WEDNESDAY, AUGUST 8, 2007 (15.10–17.30)
Poster Session 3
Poster Session 3.1: Connexins in Brain and Nervous Tissue
Poster Session 3.2: Channel Structure and Function
Poster Session 3.3: Connexins and Pannexins in Skin and Sensory Organs
Poster Session 3.4: Pharmacology of Gap Junctions
SUNDAY, AUGUST 5, 2007 (19.30–22.00)
Poster Session 1
Poster 1.1–1.19
1.1 Characterization of Gap Junctional Hemichannels In Dissociated Mouse Lens Epithelial Cells and Differentiating Fiber Cells
Lisa Ebihara, Xiaoqin Liu, and Jun-Jie Tong
Rosalind Franklin University of Medicine and Science
To understand the potential physiological and pathophysiological roles of Cx46 and Cx50 hemigap-junction channels in the lens, we have characterized the properties of native Cx hemichannels in lens epithelial and differentiating fiber cells isolated from mice using the whole cell patch clamp technique in divalent cation-free Ringer solution. The following precautionswere taken to minimize the interference from potassium channels and swelling induced chloride channels: (1) cesium was used as the main cation in the pipette solution and (2) the osmolarity of the pipette and bath solutions were carefully adjusted to reduce osmotic inbalances. Moreover, in some of the experiments, chloride was replaced by gluconate in the bath solution to eliminate outward currents due to chloride influx. Under these conditions, the primary current in the differentiating fiber cells was a multichannel current composed of large conductance channels. This current was partially closed at a holding potential of −60 or −80 mV, activated on depolarization, and had a reversal potential near 0 mV. The current was mostly suppressed in the presence of physiological concentrations of divalent cations (2 mM [Ca2+]o, 1 mM [Mg2+]o). These properties resembled those of Cx46 hemichannels expressed in Xenopus oocytes. Multichannel currents composed of large conductance channels were also frequently seen in the epithelial cells and newly differentiating fiber cells. In some cells, only a few channels were observed. These channels had a single channel conductance (265–531 pS) that fell into the same range as Cx46 and Cx50 hemichannels.
1.4 In Vivo Requirement of Cx43 for the Prevention of Glucocorticoid-Induced Osteocyte and Osteoblast Apoptosis by Bisphosphonates
Lilian Plotkin1, Kanan Vyas1, J. Ignacio Aguirre2, Robert S. Weinstein1, Stavros C. Manolagas1, and Teresita Bellido1
1University of Arkansas for Medical Sciences and 2University of Florida
Cx43 is the major gap junction protein expressed in bone cells and its ubiquitous deletion leads to embryonic osteoblast dysfunction and delayed skeletal ossification. Furthermore, Cx43 expression is required for the response of bone cells to systemic or mechanical stimuli. In particular, the anti-apoptotic effect of the bone preserving agents bisphosphonates requires Cx43 hemichannel opening and ERK activation in osteocytes and osteoblasts in vitro. To establish whether Cx43 is also required for the anti-apoptotic effects of bisphosphonates in vivo, we generated mice in which Cx43 was specifically deleted from osteocytes and osteoblasts using the Cre/LoxP technology. Mice in which the Cx43 gene is flanked by LoxP sites were mated with Cx43+/-mice expressing Cre-recombinase under the control of the human osteocalcin (OCN) promoter. This cross rendered Cx43-/flOCN-Cre mice, lacking Cx43 in osteocytes and osteoblasts and control Cx43-/fl mice. In contrast to Cx43-/fl mice, Cx43 expression was absent in osteocytes or osteoblasts in bone sections or calvaria cell cultures from Cx43-/flOCN-Cre mice, as examined by immunohistochemistry and real-time PCR. Cx43-deficient mice exhibited similar bone accrual and adult peak bone mass to control mice. Female 5-month-old mice were injected daily with saline or the bisphosphonate alendronate (2.3 umol/kg/day) for 31 days, starting 3 days prior to implanting pellets releasing 2.1 mg/kg/day of the glucocorticoid prednisolone. Alendronate prevented the increase in osteocyte and osteoblast apoptosis induced by glucocorticoids in Cx43-/fl control mice. In contrast, alendronate was ineffective in preventing apoptosis in Cx43-/flOCNCre mice. Nonetheless, alendronate abolished glucocorticoid-induced bone loss in both Cx43-/fl and Cx43-/flOCN-Cre mice, consistent with evidence that glucocorticoid-induced bone loss is due to increased bone resorption, independent of glucocorticoid actions on osteoblastic cells. These results indicate that, as occurs in vitro, Cx43 expression is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo.
1.5 b-Arrestin Interacts with Cx43 and Anchors ERKs in the Cytoplasm: A Requirement for Cx43/ERK-Mediated Anti-Apoptosis by Bisphosphonates in Osteocytes
Lilian Plotkin, Kanan Vyas, Arancha R. de Gortazar, Stavros C. Manolagas, and Teresita Bellido
University of Arkansas for Medical Sciences
The anti-fracture efficacy of bisphosphonates may be due in part to preservation of the osteocyte network resulting from inhibition of osteocyte apoptosis. This effect requires Cx43 hemichannel opening and ERK phosphorylation. However, unlike ERK-mediated survival induced by other stimuli such as 17b-estradiol, the bisphosphonate-activated Cx43/ERK pathway does not result in nuclear ERK accumulation and does not require nuclear function of ERKs. Instead, it depends on the phosphorylation of ERK cytoplasmic targets and it is abolished by forced nuclear retention of the kinases. Therefore, ERK localization outside the nucleus is indispensable for osteocyte survival by bisphosphonates. We now report that ERKs and b-arrestin (a scaffolding protein that together with clathrin mediates the internalization of G protein-coupled receptors) coimmunoprecipitated with Cx43 in osteocytic cells and that the bisphosphonate alendronate increased this association. Moreover, ERK2 fused to red fluorescent protein co-localized with Cx43 fused to green fluorescent protein outside the nucleus in untreated and alendronate-treated cells. In addition, alendronate did not induce ERK nuclear accumulation in cells expressing endogenous or transfected wild-type b-arrestin (wtARR). In contrast, alendronate induced ERK nuclear accumulation in cells expressing a dominant-negative b-arrestin mutant (dnARR) that competes with endogenous b-arrestin for binding to clathrin. Importantly, alendronate activated ERKs in dnARR-transfected cells as effectively as in cells transfected with wtARR, demonstrating that dnARR does not interfere with ERK activation. Remarkably, whereas alendronate inhibited apoptosis in cells expressing wtARR, it did not in cells expressing the dnARR mutant. On the other hand, ERK nuclear localization or anti-apoptosis induced by 17b-estradiol were not affected by either dnARR or wtARR indicating the specific cross-talk between b-arrestin and the Cx43/ERK pathway. We conclude that bisphosphonates induce the formation of a complex comprising Cx43, b-arrestin and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by these agents.
Poster Session 1.2: Pannexins and Innexins
1.10 Transient Expression of Pannexin2 Protein in Hippocampal Astrocytes Following Cerebral Ischemia/Reperfusion Injury
Federico Cicirata1, Zappalà Agata2, Li Volti Giovanni3, La Delia Francesco 2, Serapide Francesca 2, and Pillitteri Rosalia4
1University of Catania, 2Dipartimento Scienze Fisiologiche, University of Catania, 3Dipartimento di Chimica biologica, Università di Catania, and 4CNR, Catania
The study was devoted to investigate the role of the pannexin2 (Panx2) in the hippocampus after a transient ischemic insult. A polyclonal antibody for the rat Panx2 was generated in chicken and it was used to study the expression pattern of Panx2 in brain of rats submitted to ischemia/ reperfusion (I/R) injury. Rats were sacrificed after 8h, 24h, 48h, 72h and 7 and 14 days from I/R. Cells positive to Panx2 were visualized by fluorescence immunohistochemistry and counterstained with GFAP (astrocytes) and parvalbumin (Pv, interneurons). Panx2 was exclusively expressed in neuronal cells: both pyramidal neurons and interneurons. About the 85% of the Pv+ cells were also Panx2+. I/R injury resulted in a progressive depletion from 8 to 72 hours of interneurons, including both Pv+/Panx2+ cells as well as Pv+/Panx-ones, in the whole hippocampus, but particularly in CA1. Concomitantly, Panx2 was expressed in astrocytes, with a progression from 24 to 72 hours, when virtually most astrocytes were Panx2+. Such expression was grossly unmodified at 7 days and almost completely disappeared at 14 days. In conclusion, our results showed that I/R injury induced a transient expression of the Panx2 in astrocytes for about 7 days.
1.11 Innexins Form Dual Function Channels
Gerhard Dahl1, Li Bao2, Stuart Samuels1, Silviu Locovei1, Eduardo Macagno2, Kenneth Muller1
1University of Miami and 2University of California, San Diego
Calcium waves occur in both vertebrates and invertebrates in diverse phyla. Recent evidence indicates that the ATP-release channel at the root of calcium wave initiation and propagation is formed in vertebrates by pannexin 1 proteins. The innexins, known to form gap junctional channels in invertebrates, have limited sequence identity with the pannexins though these two protein families appear to have similar ancestry. This led us to assay whether innexins can also form non-junctional membrane channels by expressing two innexins of the medicinal leech in frog oocytes. We found that, in addition to gap junctions, innexins also form non-junctional membrane channels with properties similar to those formed by pannexin 1. Thus, the innexin channels could be opened at resting membrane potential by mechanical stress and by micromolar cytoplasmic calcium; they exhibited single channel conductances of about 500 pS and were closed by carbenoxolone (100 μ M). In addition we found that loss of carboxyfluorescein from the giant glial cell in the connectives of the leech nerve cord, known to express the innexins we assayed, was blocked by carbenoxolone. The innexin channels thus have qualities expected of proteins involved in the generation of calcium waves, which occur in leech glia, indicating that they may exert a dual function: in addition to their conventional role as gap junction-forming proteins, they may form physiological non-junctional membrane channels. One possible specific role of many could be as an ATP release channel. These findings support the hypothesis that once deuterostomes acquired connexins, which formed diverse gap junctions, the innexin orthologs, the pannexins, were retained in vertebrates for the non-junctional channel functions.
1.12 Tumor-Suppressive Effects of Pannexin1 in C6 Gliomas
Christian Naus1, Charles P.K. Lai1, John Bechberger1, and Roberto Bruzzone2
1University of British Columbia, 2Institut Pasteur
A family of proteins, named pannexins (Panxs), has recently been identified as the mammalian counterpart of the invertebrate gap junction proteins, innexins, and has also been shown capable of forming functional hemichannel and intercellular channels. To date, Panx1 and Panx2 mRNAs were reported to be expressed in the brain. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. We have previously demonstrated that when gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation of the glioma is reduced. In this study, we examined the possible tumor-suppressive effects of Panx1 expression in C6 cells. RT-PCR analysis revealed that C6 cells do not express any of the Panx transcripts, whereas its non-tumorigenic counterpart, rat primary astrocytes, exhibited mRNAs for all three Panxs. Upon generation of stable C6 transfectants with tagged Panx1 (myc or EGFP), we observed a flattened morphology of the Panx1-expressing cells, differing from the spindle-shaped control transfectants (with the EGFP-tagged constructs only). In addition, a unique Panx1 expression pattern locating to the Golgi apparatus and plasma membrane was observed. Moreover, Panx1 expression increased passage of small fluorescent molecules, sulforhodamine 101. Finally, we demonstrated that stable expression of Panx1 in C6 cells significantly reduced cell proliferation in monolayers, cell motility, anchorage-independent growth and in vivo tumor growth in athymic nude mice. Altogether, we conclude that the loss of Panx expression may participate in the development of C6 gliomas while restoration of Panx1 plays a tumorsuppressive role. This research is supported by the Canadian Institutes of Health Research.
1.13 Pannexin2 as a Novel Growth Regulator in C6 Gliomas
Charles Pin-Kuang Lai1, John F. Bechberger2, Roberto Bruzzone3, and Christian C. Naus2
1University of British Columbia, 2Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada, and 3HKU-Pasteur Research Centre, Hong Kong SAR, China
Pannexins (Panxs) have recently been suggested as the mammalian homologue of the invertebrate gap junction proteins, innexins, and have also been shown capable of forming both functional hemichannels and intercellular channels. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. We have previously demonstrated that when gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation of the glioma is reduced, suggesting connexins as negative growth regulators. Recently, our study demonstrated a loss of Panxs transcripts in C6 cells and a partial reversion of the transformed phenotype by Panx1 once its expression restored. In this study, we pursued the possible tumor-suppressive effects of Panx2 in C6 cells. Upon generation of stable C6 transfectants with tagged Panx2 (HA or EGFP), we observed a unique Panx2 expression pattern locating only to the cytoplasm but not to the plasma membrane, which differs greatly from that of Panx1-expressing C6 cells. As determined by cell proliferation assay and soft agar assay, a significant reduction in saturation density and anchorage-independent growth, respectively, of both tagged Panx2 transfectants was also evident when compared to the control transfectant (EGFP only). Given the difference between Panx1 and Panx2 expression patterns, we speculate that the two Panxs may elicit their tumor-suppressive functions through distinct pathways. Efforts are currently underway in examining these possibilities. Collectively, these findings indicate that the loss of Panx2 transcript may also contribute to the development of C6 gliomas while restoration of its expression, in addition to Panx1, plays a tumor-suppressive role under different mechanisms. This research is supported by the Canadian Institutes of Health Research.
Poster Session 1.3: Life Cycle of Connexins
Poster Session 1.4: Regulation of Channel Activity
1.21 A New Method for Quantitatively Measuring Gap Junctional Coupling After Fluorescent Dye Microinjection
Steve Bond and Christian Naus
University of British Columbia
Direct microinjection of small, gap junction permeable tracers into cultured cells is a common technique to assess levels of intercellular coupling. The data collected is often presented quantitatively, by measuring parameters such as distance traveled by the tracer away from the site of injection, the number of non-injected cells in the vicinity which receive tracer through coupling, and the time it takes for a tracer to migrate from one cell to another. Although these parameters can yield robust, statistically significant results when comparing cell types with moderate to large variation in coupling ability; it can be difficult and time consuming to generate a similar degree of significance when faced with more subtle variation. This can force investigators to switch to other techniques, like electrophysiology or FRAP. The loss of statistical power is generally due to physical factors like variation in cell size or density when comparing different cell types or experimental conditions, and human factors, which includes bias while taking measurements. To overcome these issues, we have developed a more robust means to quantify coupling after microinjection of fluorescent tracer. A series of fluorescent micrographs are acquired over a set period of time post-injection, each receiving an equal sub-saturation excitation exposure. The injection site is identified by co-injecting the gap junction impermeable dye rhodamine-dextran with the permeable tracer carboxyfluorescein, and is subsequently cropped out of the images with commercially available image adjustment software. A region of interest which encompasses all detectable tracer in the endpoint image is selected and applied across the entire series, then total fluorescence (minus background) is measured for each, from which a ratio is generated. This method produces a more powerful quantitative assessment of coupling then many of the standard methods currently employed.
1.22 The Yin and Yang of Cx37 and Cx43
Janis Burt, Tasha K Nelson, Jennifer S Fang, and Alexander M Simon
University of Arizona
Cx37 expression is down-regulated when Cx43 is up-regulated in the vascular endothelium. To better understand their unique contributions to vascular function, we expressed mCx37 in a communication deficient cell line (Rin) such that its effects on growth, junctional conductance (gj) and channel properties could be compared to Cx43 expressing Rin cells. Unlike Cx43, the conductance of Cx37 junctions decreased spontaneously (run-down) during dual whole-cell voltage clamp measurement. Amplitude histograms revealed a uniform distribution of single channel events (before and after rundown) ranging in amplitude from 25 to 355pS with a slight preference for ∼ 80pS events. In the presence of BIM, a PKC inhibitor that prevented run-down, the ∼ 80pS event frequency increased. While TPA, a PKC agonist, decreased the ∼ 80pS event frequency, a strong preference for another amplitude population was not revealed. However, in the combined presence of TPA and okadaic acid (OA; phosphatase inhibitor) both ∼ 80pS and ∼ 350pS populations were evident. This combined treatment also resulted in an ∼4 fold increase in gj. These data suggest that Cx37 must be phosphorylated to occupy the fully open state of the channel. In untreated Cx37-TM expressing cells (putativePKC and MAPKsites, serines 302, 328 and 275, mutated to alanine) 265pS events were prevalent and neither the ∼ 80pS or ∼ 350pS populations was induced by TPA/OA treatment. These data indicate that Cx37 channel function is oppositely regulated compared to Cx43, with PKC and MAPK activity interacting to enable the fully open state. Interestingly, unlike Cx43, Cx37 or Cx37TM expression eliminated Rin cell proliferation. Cx37 may serve a growth suppressive role in vivo as well since vascular density and recovery from hindlimb ischemic injury were enhanced in Cx37 deficient animals.
1.23 CFTR Interacts with Lipid Raft-Associated Receptor Complex to Modulate TNF-Alpha-Dependent Gap Junctional Intercellular Communication
Marc Chanson1, Dudez Tecla2, and Bruce A Stanton3
1Geneva University Hospitals, HUG-PO BOX 14, 2Geneva University Hospitals, Switzerland, and 3Dartmouth Medical School, Hanover NH, USA
TNF-alpha triggers variable effects on gap junctional intercellular communication (GJIC) in cells expressing Cx43. We have reported a defect in the regulation of GJIC by TNF-alpha in cystic fibrosis (CF) airway cells as compared to non-CF cells expressing the CF gene (CFTR). To provide a mechanism explaining this variability, we have studied GJIC in MDCKI epithelial cells expressing GFP-tagged wild-type (WtCFTR), mutant CFTR lacking its PDZ-binding C-terminal motif (CFTR-delTRL) or mutant CFTR exhibiting the delF508 mutation (delF508-CFTR), which causes CF. Localization of receptors to detergent-resistant membrane microdomains (DRMs), or lipid rafts, is thought to trigger specific intracellular signals ultimately regulating cell fate. In MDCKI cells, we found that TNF-alpha increased the proportion of WtCFTR but not CFTRdelTRL in DRMs. The TNF-alpha-dependent recruitment of wild-type CFTR to DRMs was reduced after cell surface extraction of cholesterol and modulated by tyrosine kinase activity. The amount of wild-type CFTR in DRMs correlated with the one of the tyrosine kinase c-Src. TNF-alpha and modulators of tyrosine kinase activity also recruited TNFR1 to DRMs. These TNF-alpha-mediated recruitments were not observed in CFTR-delTRL-expressing cells, suggesting that the PDZ-binding domain of CFTR confers to TNFR1 the ability to mobilize in DRMs. This possibility was confirmed by the distinct biological responses of parental MDCKI, WtCFTR, CFTR-delTRL and delF508-CFTR cells exposed to TNF-alpha. Only MDCKI cells expressing WtCFTR showed marked inhibition of GJIC. These results indicate that CFTR in association with c-Src and TNFR1 presented within the context of lipid rafts provide a responsive signaling complex at the surface of epithelial cells to trigger specific biological responses. Defect in this mechanism may alter TNF-alpha signaling in CF epithelial cells. The presence or absence of a TNFR1 signaling complex in lipid rafts may also provide a rational explanation for the variable effects of TNF-alpha on GJIC in Cx43-expressing cells.
1.24 Involvement of the Small GTPase Rho a in the Basal Permeability of Connexin43 Channels in Rat Ventricular Myocytes, a Pathway in Serotonin Signalling
Mickael Derangeon1, N. Bourmeyster1, I. Plaisance1, M. Popoff2, J. Argibay3, D. Sarrouilhe4, and J.C. Hervé1
1CNRS umr6187 Université de Poitiers, France, 2Institut Pasteur, Paris, France, 3CNRS umr 6542 Université de Tours, France, and 4Physiologie Humaine, Faculté de Médecine/Pharmacie, Poitiers, France
Gap junctions are clusters of transmembrane channels built by docking of two hemichannels or connexons, each formed by assembly of six connexins surrounding an aqueous pore. Connexin43 is the main gap junction protein expressed in neonatal rat ventricular myocytes. Recent studies suggested that RhoA mediates hypertrophic signals in cardiac myocyte hypertrophy; on the other hand, various data studies also put forward that some connexins and serotonin receptors would be involved in this cardiac disease. The aim of the present study was to examine the possible involvement of Rho GTPase in the modulation of the permeability of Cx43 channels and the potential implication of serotonin receptors in this regulation. Both the specific activation of the RhoA GTPase cascade by the Cytotoxic Necrotizing Factor 1 (CNF-1, 200 ng/ml) or a 10 min exposure to serotonin markedly enhanced the cell-to-cell diffusion of a fluorescent dye, estimated by the Fluorescence Recovery After Photobleaching (FRAP) method but their simultaneous application had no additional effects. Conversely, an irreversible Rho-A inhibitor (Clostridium botulinum C3) significantly reduced the Gap Junctional Intercellular Communication (GJIC). The serotonin effect was abolished when ketanserin, a 5-HT2a inhibitor, was simultaneously added. 5-HT2a is known to activate PKC and/or Rho-A GTPase pathways. Calphostin C, a PKC inhibitor, slightly reduced the serotonin response whereas C3 virtually abolished serotonin effects on intercellular coupling. Moreover, the Rho-A activity, measured by a pull-down approach using GST-Rhotekin, was markedly increased after serotonin stimulation. Western-blotting analysis showed that phosphorylation profiles of Cx43 remained unmodified after serotonin or C3 application. The inhibition of Rho-A downstream kinase effectors by Y27632 had no effect on GJIC. In conclusion, 5-HT2a receptor could regulate GJIC through the activation of a Rho-A pathway. Furthermore the present results showed the importance of the small G-protein Rho-A pathway in the control of the basal state of GJIC.
1.25 Serotonin Markedly Enhances Gap Junctional Intercellular Communication in Auricular Myocytes of New-Born Rat
Mickael Derangeon1, N. Defamie1, N. Bourmeyster1, V. Bozon2, J. Argibay2, D. Sarrouilhe3, and J.C. Hervé1
1CNRS umr6187 Université de Poitiers, 2CNRS umr6542 Université de Tours, France, and 3Laboratoire de Physiologie Humaine, Faculté de Médecine et de Pharmacie, Université de Poitiers, France
“G protein coupled receptors” (GPCR) form one of the largest families of the human genome and represent more than 850 genes. They control the physiological activity and the homeostasis of the majority of the cells and are the privileged target for 30 to 45% of the usually prescribed drugs. One of these GPCRs, serotonin (5-hydroxytryptamine) 4 receptor isoform (5-HT4) present in human atrial myocytes, would be involved in the development of cardiac diseases. Gap junction intercellular communication (GJIC) allows electrical and mechanical synchronisation between individual cardiac myocytes. The aim of the present study was to examine the possible interplay between serotonin receptors and the gap junctional proteins Connexin 40 (Cx40), -43 and -45 present in neonatal auricular myocytes. The functional state of connexin channels has been investigated by means of the intercellular diffusion of a fluorescent dye (6-carboxyfluorescein), quantified by the Fluorescence Recovery After Photobleaching (FRAP) technique. A 10 min exposure of auricular myocytes to serotonin markedly enhanced the intercellular coupling. Serotonin application in presence of a 5-HT4 inhibitor strongly increased the GJIC level. 5-HT2a and 5-HT2b inhibitors decreased the GJIC degree and the response was completely abolished when a 5-HT4 inhibitor was added. Western-blotting analysis showed that phosphorylation profiles of Cx40, -43 and -45 remained unmodified after serotonin application. Moreover, Real Time RT-PCR and western blotting showed that a prolonged exposure to serotonin strongly decreased the expression level of Cx40 as well as a weak reduction in Cx43 and -45 expressions. In conclusion, serotonin, via 5-HT2 and 5-HT4 receptors, had a dual action on the strength of intercellular communication between neonatal auricular myocytes, a short-term enhancing effect on GJIC as well as a genomic effect on Cx expression when the exposure was prolonged.
1.26 Src Utilizes Cas to Block Gap Junctional Communication Mediated by Connexin43
Gary Goldberg1, Yongquan Shen1, P Raaj Khusial1, Xun Li1, Hitoshi Ichikawa2, and Alonso P Moreno3
1UMDNJ, 2NCCRI, 3Cardiovascular Research and Training Institute, University of Utah
Malignant and metastatic tumor cells often display less gap junctional communication than their benign or normal counterparts. The Src tyrosine kinase phosphorylates Cas to confer metastatic growth potential to transformed cells. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src utilizes Cas to close gap junction channels formed by Cx43. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.
1.27 A Mathematical Model of Intercellular Calcium Wave Spread and Alteration in Disease
Dumitru Iacobas, David Spray, Eliana Scemes, Sylvia Suadicani
Albert Einstein College of Medicine
We describe a 2D stochastic model of intercellular Ca2+ waves (ICWs) that includes contributions of external stimuli, ionotropic and metabotropic receptors, exo- and ectonucleotidases, second messengers and gap junctional communication. In this model, an initial stimulus evokes ATP and UTP release from a single cell. Agonists diffuse and are degraded both in bulk solution and at cell surfaces. Ca2+ elevation in individual cells is determined by bound agonist concentrations and by number and features of P2 and glutamate receptors summed with that generated by IP3 diffusing through gap junction channels. Variability of ICWs is provided by randomly distributing a predetermined density of cells in a rectangular grid and by randomly selecting within intervals values characterizing the extracellular compartment, individual cells and interconnections with neighboring cells. Variability intervals were obtained from experiments on muscle cells from bladder and corpora of normal and STZ-induced diabetic mice. The simulation program permits individual change of ICW components, allowing comparison of simulations in normal and diabetic muscle. Such modeling is expected to be useful for testing phenomenological hypotheses and in understanding consequences of alteration of system components under experimental or pathological conditions.
1.28 The Role of Ras, but not Stat3, Signaling Critical for Suppression of Gap Junctional Intercellular Communication in v-Src Transformed Cells
Satoko Ito, Michinari Hamaguchi, and Takeshi Senga
Nagoya University Graduate School of Medicine
Background: Cell transformation by v-Src causes suppression of gap junctional intercellular communication (GJIC). Although tyrosine phophorylation of connexin43 (Cx43), a gap junctional component, seems to be necessary for the suppression, involvement of other signaling activated by v-Src remains unclear. We investigated the role of Ras, MEK and Stat3 signalings in the suppression of GJIC by v-Src.
Methods: Rat fibroblast cell line 3Y1 and 3Y1 transformed with v-Src (SR3Y1) were used. To measure GJIC, the number of Lucifer Yellow dye coupled cells was counted and monitored by fluorescent microscopy after the microinjection of dye into single cell.
Results: Conditional expression of S17N Ras, which acts as dominant negative Ras, dramatically recovered GJIC in v-Src transformed cells. Conditional expression of mtGAP1m, which is constitutive active mutant of GAP for Ras, also recovered GJIC in v-Src transformed cells. Although expression of S17N Ras or mtGAP1m substantially decreased the levels of active Ras, tyrosine phosphorylation of cellular proteins including Cx43 remaind unchanged. Similarly, treatment of v-Src transformed cells with a Ras farnesyltransferase inhibitor, manumysin A, restored GJIC, whereas tyrosine phophorylation of Cx43 remained unchanged. In contrast to Ras signaling, treatment with siRNA against Stat3 or MEK inhibitor, U0126, could not recover GJIC in v-Src transformed cells. In addition, expression of constitutive active MEK in 3Y1 did not suppress but increased GJIC.
Conclusion: These results strongly suggest that, in addition to Cx43 phosphorylation, constitutive activation of Ras signaling is required for the suppression of GJIC by v-Src. In contrast, Stat3 and MEK-MAP kinase signalings are not critical for the suppression of GJIC by v-Src.
1.29 The Effect of Modified Connexin Expression Profiles on Cell Cycle Progression
Scott Johnstone1, Patricia Martin1, and Rachel Errington2
1Glasgow Caledonian University and 2University of Wales College of Medicine
Connexin (Cx) mediated communication (CMC) is believed to be important in cell proliferation, migration and differentiation. Cx over-expression has been used to examine the role of CMC in tumourgenesis and changes in Cx43 and Cx26 expression are associated with wound healing events making these proteins attractive therapeutic targets. In the present study we have investigated the effect of differentially regulated Cx43 expression and functionality, using timelapse microscopy and FACS analysis to dissect changes that occur in cell cycle traverse under differing levels of Cx43 expression. The results indicate that high levels of Cx43 expression induce a cell cycle stall in G1. HeLa cells, selected to express Cx43 and primary human fibroblasts (that predominantly express Cx43) were used in these studies. Low doses, (0.5mM) of the histone deactelyase inhibitors Sodium Butyrate and Tolbutamide and the anti-arrhythmetic peptide AAP10 (50nM), were used to upregulate Cx43 expression. All three agents enhanced Cx43 expression and functionality following 24hr treatment as determined by immunoflouresence, western blot and the ability of cells to transfer small fluorescent dyes following microinjection. GJIC was blocked by treating cells with 18aga. 24hr time-lapse microscopy was performed and mitotic and inter-mitotic durations of the cells under differing conditions were extracted. Images were captured every 5mins and collated using Metamorph software. Highly quantitative data was produced using FACS analysis of the cell cycle following treatment with the G2 cell cycle blocker Colcemid. The results showed that upregulated Cx43 expression correlated with enhanced GJIC and was associated with increases in mitotic duration, and elevated levels of p21 expression linked to a stall in G1. By contrast blocking of GJIC via treatment of the cells with 18aga had no effect on mitotic outcome. Our data adds strength to the argument that Cx43 expression and signaling are important in cell cycle mediated events.
1.32 Cytoskeleton Remodelling Induced by Sphingosine 1-Phosphate Controls Myogenesis Through the Regulation of Cx43-Formed Gap Junctions
Elisabetta Meacci1, Martinesi Maria2, Sassoli Chiara3, Squecco Roberta4, Zecchi-Orlandini Sandra3, Formigli Lucia3, and Francini Fabio4
1University of Florence; 2Departments of Biochemical Sciences, 3Departments of Anatomy, Histology and Forensic Medicine, and 4Departments of Physiological Sciences, University of Florence
Introduction. Sphingosine 1-phosphate (S1P), a lipid mediator originated by sphingomyelin degradation, is capable to regulate a wide variety of important cellular functions. Although, the bioactive lipid can act as an anti-proliferative and a pro-differentiating agent in C2C12 myoblasts, its downstream effectors are poorly known. Recently, we have demonstrated that the induction of myogenesis by S1P required the activation of several signaling pathways, involving the upregulation of Cx43 protein expression via a calcium and p38 MAPK-dependent cascades, the formation of stress fibers (SF) and, the opening of mechano-sensitive voltage-independent channels named stretch-activated channels (SACs). Specific objectives. Despite all these observations, the involvement of cytoskeleton and mechano-transduction on the regulation of Cx43-formed gap junction in skeletal muscle cells remains to be elucidated. Results. To this purpose, in the present study, we examined the role of actin remodelling and SAC-mediated cation current on Cx43 expression and gap junction functionality employing a pharmacological approach. Interestingly, we found that dihydrocytochalasin B, a selective actin disrupter agent, as well as gadolinium chloride, a specific pharmacological inhibitor of SACs, prevented S1P-induced Cx43 protein expression and gap junction intercellular communication. These data were also confirmed using updated molecular cell biology methodologies such as RNA interference and overexpression of wild type or catalytically inactive specific proteins. Conclusions. Overall, these results sugggest that, in myoblasts stimulated with S1P, formation of SF and the following activation of transmembrane cation currents through SACs are essential steps in the regulation of Cx43 expression and its assembly into functional gap junctions. In addition, these findings provide novel information regarding the mechanisms by which F-actin reorganization and mechano-transduction may regulate skeletal myogenesis. This work was supported in part by funds from University of Florence (ex-60%) to E.M., S.Z-O, F.F. and Ente Cassa di Risparmio di Pistoia e Pescia to E.M.
1.33 Studies on the Phosphorylation of the Extreme C-Terminal Tail of Connexin 43
Svein-Ole Mikalsen1, Rune Sundset2, Kirsti Ytrehus2, and Véronique Cruciani1
1Department Cancer Prevention, Inst. for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, Montebello, Oslo, Norway and 2Department of Medical Physiology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway
Connexin43 (Cx43) is the target for many kinases, e.g., Src, Erk, CKI and PKC. Several members of the PKC family may phosphorylate Cx43, depending on the system studied. It was shown that PKC phosphorylated Cx43 at serine368 (S368). A phosphospecific antibody recognizing phosphoserine368 (anti-pS368) is available (Chemicon and Cell Signaling). By using this antibody, it was shown that phosphorylation of Cx43 varies during cell cycle, wound healing of the human skin, and differentiation and development. Interestingly, the antibody 13-8300 (Zymed) is claimed to recognize the unphosphorylated S368. Thus, the two antibodies should have complementary activities. We first investigated the recognition of Cx43 by anti-pS368 using rat NRK, Syrian hamster SHE, and Chinese hamster V79 cells. There were obvious system-specific differences in anti-pS368 recognition for both control cell cultures and cultures exposed to the PKC activator, tetradecanoylphorbol-acetate (TPA). We then immunoprecipitated Cx43 from TPA-exposed NRK and V79 cells, and control V79 cells, using anti-pS368. The immunoprecipitates were probed with anti-panCx43 (Sigma C6219), anti-pS368 and 13-8300. While the recognition pattern was as expected for anti-pS368, anti-panCx43 detected three phosphovariants in NRK and V79 cells, and 13-8300 detected bands from control and TPA-exposed V79 cells. These results suggest that several phosphovariants of Cx43 may combine into a connexon. They may further suggest that the V79 connexons contain unphosphorylated Cx43, or, alternatively, the same Cx43 variant is recognized by both anti-pS368 and 13-8300, which should not occur if they had complementary recognition. The lack of complementarity is strengthened by a simple argument: none of the two antibodies recognize P1 or P2 variants of Cx43. We have therefore performed competition experiments with synthetic phosphopeptides. The initial results suggest that the recognition patterns are not as simple as believed, in that both anti-pS368 and 13-8300 may have alternative recognition abilities.
1.40 Transjunctional KCl Gradients Alter the Vj-Dependent Gating and Spermine Inhibition of Connexin40 Gap Junctions
Richard Veenstra and Xianming Lin
SUNY Upstate Medical University
Spermine inhibits rat connexin40 (Cx40) gap junctions. Glutamate residues at positions 9 and 13 and a basic amino acid (HKH) motif at position 15-17 on the amino terminal (NT) domain are essential for this inhibitory activity. Questions remain as to whether spermine occludes the channel within the ion permeation pathway. To examine this question, cis or trans [KCl] was systematically lowered and the equilibrium dissociation constants (Kd) and kinetics of unilateral spermine block on wild-type Cx40 gap junctions were determined. Asymmetric reductions in the trans [KCl] produced noticeable asymmetric shifts in the V1/2 and Gmin values that progressively resembled Gj-Vj relationships observed in heterotypic connexin gap junction combinations. As cis or trans [KCl] was reduced by 25, 50, or 75% relative to the spermine-containing side, the transjunctional voltage (Vj)-dependent Kd values increased or decreased respectively. The spermine on-rates and off-rates, calculated from the junctional current decay and recovery time constants, were similarly affected. Hill coefficients for the spermine dose-response curves were approximately 0.58, indicative of negative cooperativity and possible multiple spermine inhibitory sites. The equivalent “electrical distance” (δ) ranged from 0.61 at 25% cis [KCl] to 1.4 at 25% trans [KCl] with a Hill coefficient of 1.0. Symmetrical reductions in [KCl] resulted in intermediate decreases in the spermine Kds, indicative of a minor electrostatic effect and a more significant effect of the transjunctional KCl electrodiffusion potential on the spermine association and dissociation rates. These data are consistent with a single spermine molecule being sufficient to occlude the Cx40 gap junction channel within the KCl permeation pathway.
1.41 PDGF-Activated Regulation of Connexin43 Gap Junction Selectivity
Jose Francisco Ek Vitorin, Timothy D. Steele, Tasha K. Nelson, and Janis M. Burt
University of Arizona
In Cx43 expressing cells, non-linearity between the rate of transjunctional dye diffusion (kNBD; cationic dye with limiting diameter of ∼ 5 A°, NBDM-TMA) and junctional conductance (gj) was previously reported. Moreover, junctions in which subconductive (55–70 pS) channels were detected had higher permselectivity (kdye/gj, min-1nS-1) than junctions in which such conductive states were not detected. Lack of correlation between permselectivity and gj could bear physiological significance if it allowed for selective metabolic coupling in the face of decreasing macroscopic dye (pj) and electrical coupling, as occurs in response to injury, disease, or growth stimulus. PDGF stimulation consistently induces phosphorylation of Cx43 and decreases in pj and gj, as well as channel conductance (yj). By measuring kdye (min-1) and gj (nS) as a function of time following PDGF (10 ng/mL) application, we show here that despite PDGF induced decreases in gj (> 50%) and pj, permselectivity was increased by PDGF compared to untreated pairs (control: 0.082 ± 0.015, n = 34; PDGF: 0.220 ± 0.060, n = 16; p < 0.05). Junctions composed of mutant Cx43-S368A, which display lowpermselectivity under control conditions, showed no significant PDGF induced change in this parameter (control: 0.014 ± 0.003, n = 16; PDGF: 0.017 ± 0.006, n = 6) despite a modest shift of γ j towards 80 pS. These data suggest that PDGF induces by phosphorylation at S368 a channel configuration with high dye permeability. The overall high junctional permselectivity induced by PDGF may constitute a regulatory trigger within tissues that primes the cells for subsequent growth responses.
1.42 Connexin Mimetic Peptides: Connexin Specificity and Effects on Human Epidermal Fibroblast Wound Closure
Catherine Wright, Malcolm Hodgins, and Patricia Martin
Glasgow Caledonian University
Manipulation of connexin mediated communication networks can modify wound healing rates by altering epidermal keratinocyte and or fibroblast behaviour. Agents that directly target specific connexins have great therapeutic potential. To this end we analysed the connexin (Cx) specificity and effects of three connexin mimetic peptides, targeted to the extracellular loops of Cx43, on cell-cell communication and cell proliferation and migration responses during fibroblast wound healing, with a view to elucidate their therapeutic potential in regulating wound healing events. To determine peptide specificity HeLa cells stably transfected to express Cx43, Cx26 or Cx40 were used. Primary human skin fibroblasts were also cultured and used in cell migration assays. Following treatment with connexin mimetic peptides (Gap26, Gap26M and Gap27) cells were microinjected with Alexa488 (MW 570 Da) or Alexa594 (MW 795 Da); the extent of dye spread between neighbouring cells was monitored. RTPCR determined the connexin expression profile of the fibroblasts, and migration assays compared rates of fibroblast wound closure with and without peptide. Gap26 and Gap26M dose-dependently inhibited dye transfer in all three HeLa cell model systems i.e. in a non-connexin specific manner (ANOVA: n = 250; P < 0.01), whereas Gap27 was more effective in HeLa-Cx43 cells than in cells expressing Cx26 or Cx40 at low concentrations (50 μ M). Gap26M significantly reduced dye transfer between fibroblasts at 10 μ M-100 μ M (n = 250; P < 0.01). Fibroblasts expressed Cx43, Cx40 and Cx45 mRNA and treatment with Gap26M did not alter Cx expression levels. In migration assays 100 μ M Gap26M increased wound closure rates in both mouse keratinocytes and human fibroblasts. In conclusion, Gap26 and Gap26M are non-connexin-specific peptides where as Gap27 is Cx43 specific. Gap26M and Gap27 were also effective at low doses (10–50 μ M). Gap26M effectively blocked cell-cell communication in primary fibroblast cells and increased wound closure rates, exemplifying the potential of connexin mimetic peptides as therapeutic agents.
1.43 Analysis of Transgenic Mice Overexpressing Cx43 in Adult Skeletal Muscle
Volker Wulf1, Julia von Maltzahn1, Daniela Wenzel2, Philipp Sasse2, Britta Döring1, Bernd Fleischmann1,2, and Klaus Willecke1
1Institut für Genetik, and 2Institut für Physiologie I, Universität Bonn, 53105 Bonn, Germany
Connexin43 (Cx43) is known to be expressed during myogenesis and muscle regeneration. In contrast, adult skeletal muscle is one of the few tissues lacking connexin expression. The electrical isolation of each fiber is considered to be crucial for proper muscular functions. To address this issue we generated mice overexpressing Cx43 the and enhanced green fluorescence protein (eGFP) in skeletal muscle. For this purpose we used the Tet-Off system controlled by the skeletal muscle specific muscle creatin kinase (MCK) promotor driving the bidirectional transcription of Cx43 and eGFP. Transgenic mice showed no histological alterations but immunoblot and immunofluorescence analyses of embryonic and adult tissue confirmed skeletal muscle specific overexpression of Cx43 and eGFP. Only the P0 (“non phosphorylated”) protein band of overexpressed Cx43 could be detected by immunoblot in adult skeletal muscle. Furthermore, immunofluorescence analysis showed Cx43 immunosignals in the plasma membranes suggesting gap junctional coupling between adjacent myofibers. In addition, isometric contraction studies of fast and slow twitch skeletal muscles (extensor digitorum longus and soleus), yielded normalized force-frequency relationships displaying higher relative forces at given frequencies for Cx43 overexpressing muscles. Nevertheless, functional coupling between myofibers could not be confirmed so far by microinjection of adult skeletal muscle using different fluorescent dyes. More experiments will be performed in different muscle types and preparations to investigate this in more detail. At this stage, the lack of gap junctional coupling in dye-injection experiments may be attributed to a mechanical boundary provided by the endomysium ensheating each fiber, improper phosphorylation or sustained dephosphorylation leading to closed Cx43 channels. The obtained results of the contraction studies might be explained by very low level of Cx43 mediated gap junctional coupling between muscle fibers or due to non gap junctional effects of overexpressed Cx43 in skeletal muscle.
MONDAY, AUGUST 6, 2007 (20.00–22.30)
Poster Session 2
Posters 2.1–2.17
Poster Session 2.1: Connexins in the Cardiovascular System
2.5 Electrical Properties of Heteromeric-Homotypic Channels Made of Cardiac Tandem Connexins Cx40, Cx43 and Cx45
T. Desplantez1, N. Thomas2, N.J. Severs2, R. Weingart1, and E. Dupont2
1Department of Physiology, University of Bern, Bern, Switzerland and 2NHLI, Imperial College London, London, UK
Cardiac muscle cells express three main connexin (Cx) isoforms, Cx40, Cx43, Cx45. Tissue-specific patterns of Cx expression raises the possibility that heteromeric connexons may form. While several studies in the past have indicated that Cx40, Cx43 and Cx45 have the capacity of forming heteromeric connexons and gap junction channels (GJC), so far no data are available on the electrical properties of heteromeric GJC of known Cx composition. In order to address this problem, we have made tandem-Cx constructs. cDNA of Cx40, Cx43 and Cx45 was pairwise linked (Leu-Glu di-peptide linkers) via amino and carboxy terminal (Cx1-Cx2). Nine constructs have been engineered, Cx1 and Cx2 being one or the other Cx, and transfected into HeLa cells. Two years ago, we presented data on the electrical properties of homomeric-homotypic GJC made of the fusion proteins Cx40-Cx40, Cx43-Cx43 and Cx45-Cx45. Now we present results obtained from heteromeric-homotypic GJC consisting of the fusion proteins Cx40-Cx43, Cx43-Cx40, Cx40-Cx45, Cx45-Cx40, Cx45-Cx43 and Cx43-Cx45, using a voltage-clamp method in the whole cell configuration. We found that each tandem construct forms functional GJC. At the multichannel level, the currents Ij inactivate with time. At steady state, they can be described with the Boltzmann equation (half-maximal inactivation Vj0, minimum conductance Gjmin, gating charge z), kinetics of Ij inactivation. At the single channel level, Ij exhibits a main and a residual conductance state (γ jr and γ jm). Interestingly, tandems exhibit different properties depending on the order of the Cxs in the tandem. Conceivably, this reflects specific role for the amino and the carboxy terminal domains. These observations suggest the formation of heteromeric homotypic channels by these connexins in cardiac tissue. In further experiments we will elucidate the properties of channels by varying the ratio of co-expressed connexins, and their specific functions in the cardiac action potential propagation will be determined.
2.8 Does Increased Expression of Connexin45, as Found in Heart Failure, Contribute to Decreased Gap Junction Size?
Katharina Grikscheit, Alexandra Bruce, Emmanuel Dpont, Neil Thomas, and Nicholas J. Severs
National Heart and Lung Institute, Imperial College London, UK
In the human heart, working ventricular myocytes express connexin43 (Cx43) and traces of connexin45 (Cx45). In congestive heart failure, Cx43 levels decrease, whereas Cx45 levels are reported to increase. Heart failure is also accompanied by a decrease in gap junction size. These findings raise the possibility that alterations of the connexin co-expression ratio may regulate gap junction size. As this hypothesis cannot be investigated in human tissue, we have engineered an RLE cell line to express inducible, graded levels of Cx45 under stimulation of the insect hormone ponasteroneA, in addition to constant levels of endogenous Cx43. Gap junction sizes were measured in immunoconfocal images of induced or non-induced cells immunolabelled for either Cx43 or Cx45. In induced cells (i.e. those expressing Cx45), gap junction sizes are significantly smaller than those of non-induced cells (non-induced 0.66 μ m ± 0.012 μ m, N = 850 versus induced 0.59 μ m ± 0.01 μ m, N = 791; reduction 11%; P < 0.0001), irrespective of whether the junctions are labelled for Cx43 or Cx45. Using the Triton X-100 solubility assay, quantitative analysis of the total connexin ratio in junctional versus non-junctional fractions demonstrates that the total number of junctional, docked connexons (both Cx43 and Cx45) increases approximately two-fold at maximum induction. These results suggest that cells expressing Cx45 in addition to Cx43 may have more numerous, but smaller gap junctions. Double immunolabelling for Cx43 and Cx45 shows co-localization at gap junctions, and preliminary immunoprecipitation experiments indicate that Cx43 and Cx45 do indeed interact. Taken together, these results suggest that gap junction size may be regulated by the stoichiometry of connexin isoforms within the connexon and/or the gap junction, and specifically that Cx45 in the failing heart may contribute to decreased gap junction size, but not to an overall decrease in Cx43 gap junctions.
2.9 Phosphatidylinositol-Bisphosphate Regulates Intercellular Coupling in Cardiac Myocytes
Johannes P. Hofgaard,1 Kathrin Banach,2 Sarah Mollerup,1 Helene Korvenius Jørgensen,1 Søren-Peter Olesen,1 Niels-Henrik Holstein-Rathlou1, and Morten Schak Nielsen1
1The Danish National Research Foundation Centre for Cardiac Arrhythmias and Department of Biomedical Sciences, Panum Institute, University of Copenhagen and 2Department of Cellular and Molecular Physiology, Stricht School of Medicine, Loyola University Chicago
Aims: Phosphatidylinositol-(4,5)bisphosphate (PIP2) levels in the plasma membrane regulate many channels and transporters relevant to cardiac tissue. Whether PIP2 regulates gap junctional intercellular coupling (GJIC) in cardiomyocytes is unknown, but could add to the risk of arrhythmia during stimulation of Gα q-coupled receptors. Hypothesis: PIP2 regulates GJIC and stimulation with Gα q-coupled agonists reduces GJIC partly due to a reduction of PIP2. Methods: GJIC was measured by dye transfer after localized electroporation of Lucifer Yellow in cultured cardiomyocytes from neonatal rats. Conduction velocity (CV) was measured in cardiomyocytes grown on electrode arrays. Results: One hour wortmannin exposure reduced GJIC indicating that PIP2-depletion inhibits GJIC. In contrast, hypertonic shock, which increases PIP2, increased GJIC. GJIC was inhibited by AngII and noradrenaline stimulation. To test if the reduction in GJIC after agonist stimulation was caused by PIP2-depletion; myocytes were stimulated by AngII and then allowed to recover in control medium with or without wortmannin. GJIC fully recovered in control medium whereas no recovery occurred in the presence of wortmannin. Inhibition of PKC did not affect the response to either AngII or noradrenaline. Also inhibition of arachidonic acid production did not affect the response to agonist stimulation. In beating myocytes CV was reduced by AngII stimulation. After wash-out, CV recovered and this recovery was prevented by inhibition of PIP2 production. Conclusion: Reductions in PIP2 inhibits GJIC in cardiomyocytes, and stimulation by physiologically relevant agonists can reduce PIP2 and thereby GJIC by this mechanism. This reduction lowered CV, which could lead to increased susceptibility to arrhythmias.
2.10 Oxidized Lipids Induce Spatial and Temporal Effects on Gap Junction-Mediated Calcium Signaling Between Vascular Cells
Brant Isakson1, Paul Lampe2, and Norbert Leitinger1
1University of Virginia, Cardiovascular Research Center and 2Fred Hutchinson Cancer Research Center
Heterocellular calcium communication between endothelial cells (EC) and smooth muscle cells (SMC) through gap junctions at myoendothelial junctions (MEJ) is essential to vascular function. Using a vascular cell co-culture (VCCC) composed of spatially segregated monolayers of EC and SMC that allow gap junction formation at MEJs (composed of Cx40 and Cx43), individual cell types can be stimulated and the effect on the unstimulated cell type analyzed. For example, phenylephrine (PE) applied to SMC induces an increase in intracellular Ca2+ concentrations ([Ca2+]i) first in SMC, followed by [Ca2+]i increases in EC. When ATP is added to EC, [Ca2+]i increases occurs first in EC, then in SMC. In both cases, the increase in [Ca2+]i in the unstimulated cell type is blocked by gap junction inhibitors. We have demonstrated that the proinflammatory oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) disrupts heterocellular communication. Two components of OxPAPC; 1-palmitoyl-2-glutaroylsn-glycero-3-phosphorylcholine (PGPC) and 1palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (PoVPC) were shown to upregulate PKC and PKA, respectively; both kinases induce connexin phosphorylation. Using the VCCC, we show that pretreatment of EC with PGPC (15 μg) for 20 minutes inhibited EC-induced increase in [Ca2+]i in the SMC and pre-treatment of SMC with PGPC for 20 minutes inhibited the SMC-induced increase in EC [Ca2+]i. PoVPC (15 μg) had no effect. However, when PGPC was added to EC for 20 minutes, and then PoVPC was added for another 20 minutes, heterocellular calcium communication to SMC was restored. PoVPC also restored heterocellular calcium communication to EC when SMC were treated with this protocol. Conversely, pre-treatment with PoVPC on EC or SMC for 20 minutes, and replacement with PGPC for another 20 minutes inhibited all heterocellular calcium communication. We conclude that PGPC and PoVPC spatially and temporally control heterocellular calcium communication in the vasculature, possibly due to PKC or PKA mediated connexin phosphorylation at the MEJ.
2.11 Gap Junctions in the Onset of Arterial Vasomotion
Jens Christian Brings Jacobsen1, Christian Aalkjær2, Holger Nilsson2, Vladimir Matchkov2, Jacob Freiberg3, and Niels-Henrik Holstein-Rathlou1
1Institute of Biomedical Sciences, University of Copenhagen, 2University of Aarhus, and 3University of Copenhagen
Vasomotion is a rhythmic contraction of small blood vessels. It has been found in the microvasculature in numerous species including humans. The physiological role of vasomotion is not known and the cellular mechanisms underlying its initiation are only partly understood. We present a mathematical model of a syncytium of cells. Each cell has three compartments; SR, cytoplasm and plasma-membrane and couples to its six nearest neighbors through gap junctions. Gap junctions are modeled as simple intercellular pores with a certain permeability for each ion. The SR/cytoplasm system shows autonomous calcium oscillations due to calcium-induced calcium-release (CICR) from the SR. These oscillations are modified by calcium fluxes across the plasma membrane. A cGMP-sensitive chloride channel in the membrane causes depolarization in response to calcium release from the SR, but only if [cGMP] is sufficiently high. Depolarization in turn causes uniform influx of calcium through L-type calcium channels, stimulating CICR from the SR. Results: At low [cGMP] unsynchronized calcium waves are seen in the individual cells. If [cGMP] is increased, activation of the chloride channel causes transition from low frequency waves to high-frequency whole-cell oscillations and abrupt intercellular synchronization. Similar transition and synchronization is seen experimentally at the onset of vasomotion. Intercellular synchronization occurs as a certain minimum level of gap-junction coupling is exceeded. Once synchronization is established the oscillation frequency does not change with increased intercellular coupling. Flow of calcium through gap-junctions is not necessary to achieve synchronization which occurs readily under selective gap-junction blockade of calcium. Rather, cellular entrainment is caused by intercellular equilibration of the membrane potential, uniform calcium influx through L-type calcium channels and thereby synchronization of CICR in all cells. Conclusion: The model can explain several key observations related to vasomotion in mesenteric arteries. These include transition from waves to whole-cell oscillations, increase in oscillation frequency and abrupt intercellular synchronization.
2.12 Deletion of Connexin 40 Strongly Alters the Function of Renin Producing Cells, Causing Dislocation of the Cells, Renin Hypersecretion and Hypertension
Armin Kurtz1, Charlotte Wagner2, Frank Schweda1, Lisa Kurtz1, and Cor de Wit2
1Institute of Physiology, University of Regensburg, Germany and 2Institute of Physiology, University of Lübeck
The aspartyl-protease renin is the central control factor of the so-called renin-angiotensin-aldosterone system (RAAS), which is a main regulator of volume and blood pressure homeostasis in the body. Renin is predominantly produced in the kidney. In the adult kidney renin producing cells are typically located in the walls of afferent arterioles at the transition into the glomerular capillary network. The mechanisms responsible for restricting renin expression to the juxtaglomerular position are largely unknown. Renin producing cells are known to form gap junction with their neighboured cells, such as vascular endothelial and extraglomerular mesangial cells. Cx40 is a main connexin in these cells. We now found that in mice lacking connexin 40, renin producing cells are absent in the vessel walls and instead are found in the extraglomerular mesangium, glomerular tuft and periglomerular interstitium. Renal renin synthesis and secretion are normally stimulated by the sympathetic nerves (SN) and are inhibited by angiotensin II (ANGII), salt overload, blood pressure (BP) and the macula densa mechanism (MD). In mice lacking Cx40 regulation of renin by SN and by salt are normal, whilst the physiological inhibitions by ANGII, BP and MD are either markedly attenuated. As a consequence, circulating renin levels in Cx40 deficient mice are strongly elevated causing hypertension. Since the characteristic inhibition of renin secretion by ANGII, BP and MD can also be attenuated by non selective gap junction blockers, we speculate that both an impairment of intercellular communication and a dislocation of cells contribute to the exaggerated secretion of renin in Cx40 deficient mice. In summary, our findings indicate, that Cx40 is very important for the “homing” of renin producing cells in the kidney and for the physiologic control of their function.
2.16 Endothelial Cell Gap Junctions in Aorta of Borderline and Spontaneously Hypertensive Rats
Ludmila Okruhlicova1, K. Dlugosova1, M. Fialova1, and I. Bernatova2
1Institute for Heart Research, SAS, Bratislava, Slovakia and 2Institute of Normal and Pathological Physiology, SAS, Bratislava, Slovakia
Vascular endothelial cells are connected by gap junctions, which facilitate the propagation of electrical and molecular signals along the vessel wall. The aim of this study was to investigate integrity of inter-endothelial junctions as well as expression of the gap junction protein connexin43 (Cx43) in aortic endothelial cells in two different models of hypertensive rats—spontaneously hypertensive rats (SHR) and borderline hypertensive rats (offspring of normotensive Wistar mothers and SHR fathers, BHR). Both, SHR and BHR exhibited significant blood pressure increase (183 ± 3 mm/Hg and 136 ± 1 mm/Hg) when compared to controls (121 ± 2 mm/Hg). Tissue of thoracic aorta of 3-monthold male SHR, BHR and age-matched normotensive Wistar rats (W) were processed for: transmission electron microscopic examination, immunofluorescent microscopy of endothelial gap junction protein Cx43 using monoclonal mouse anti-Cx43 antibody (Chemicon), and Western blot analysis. Electron microscopy revealed focal heterogeneous subcellular alterations of intercellular junctions, particularly tight junction discontinuity in both models of hypertensive rats. In parallel, there was a decrease in endothelial Cx43 immunofluorescent signal in SHR and to lesser extent in BHR aorta compared to normotensive rats. Western blotting analysis showed suppressed Cx43 expression in aorta of BHR and significantly reduced in SHR. These results indicate that even borderline (mild) hypertension might induce intercellular junctions alterations of aortic endothelial cells and consequently impairment of intercellular communication contributing to endothelial cell dysfunction. The work was supported by VEGA and APVV grants.
2.17 Vascular Remodeling: Functional Consequences of Altered Connexin Expression
Edward Parkin, David T. Kurjiaka, Nathaneal S. Heyman, Jos'e Ek-Vitorin, Alex M. Simon, and Janis M. Burt
University of Arizona, Tucson AZ, USA
Connexin (Cx) expression in the endothelial and medial layers of the vessel wall changes during development and as a consequence of injury and disease. The mechanistic basis for, and functional consequences of, altered Cx expression in the vessel wall remain uncertain. To explore these issues we have implemented a coarctation model that results in localized hemodynamic changes without inducing disease, vascular injury, or systemic hemodynamic effects. The abdominal aortas of 3-6 month old adult mice were surgically coarcted, distal to the renal arteries, using a U-shaped clip (0.2 mm gap). Within the clipped region, which encompasses ∼1.5 mm of vessel length, the vessel assumes a rectangular cross-section (∼200 um × 900 um, O.D. with rounded ends) wherein wall strain is reduced but shear stress increased. Carotid artery blood pressure was not affected by clip placement (n = 2) and was unchanged (n = 1) 8 days later when the vessel was harvested for further study. Coarctation-induced remodeling of the vessel wall included increased expression of Cx43 within, and at the boundaries of, the clipped region in the endothelium, as well as reorientation of the endothelial cells up- and downstream of the coarcted region. The impact of these coarctation-induced changes in vascular Cx expression on cell-cell coupling and junctional selectivity is being explored using a dual dye injection technique; wherein two fluorescent dyes of similar size but opposite charge are introduced to the same cell in the endothelial layer of a live aorta, and their spread throughout the monolayer quantified. It is anticipated that the selectivity of intercellular communication in the endothelial layer will change from cation selective, reflecting high Cx37 and Cx40 expression, to a more non-selective profile as Cx43 expression increases.
2.18 Heterogeneous Ventricular Chamber Response to Gap Junction Blockade
Steven Poelzing and Rengasayee Veeraraghavan
University of Utah, Cardiovascular Research and Training Institute
Introduction: We previously demonstrated that cardiac sodium channel expression is reduced by 20% in right ventricle (RV) compared to left (LV). Paradoxically, transverse conduction velocity (CV) is greater in the right ventricle compared to the left. While reduced inward rectifier potassium current in the right ventricle may in part underlie the paradoxical increase in RV CV, the degree of CV increase is inconsistent with computer models. Therefore, we hypothesized that cell size or gap junction function underlies the paradoxical increase in RV CV. Methods: Cell size was measured from myocytes isolated from the RV or LV of guinea pig (n = 3). CV heterogeneities were quantified by optical voltage mapping during LV or RV pacing with and without the relatively specific gap junction uncoupler carbenoxolone (10 microM) Results: RV myocytes are significantly longer (126 ± 2 micrometer) than LV myocytes (119 ± 3micrometer, p < 0.05). There was no significant difference between RV and LV myocyte width (15 ± 1 micrometer). Cellular anisotropy was significantly different between the RV (8.2 ± 0.3) and LV (6.8 ± 0.2, p < 0.05). CV anisotropy in the RV is significantly lower (1.98 ± 0.29) than in LV (2.52 ± 0.29, p < 0.01), which is paradoxical to cell geometry. Carbenoxolone significantly reduced LV longitudinal and transverse CV to a similar extent (approximately 17%) such that anisotropy was not significantly different (2.60 ± 0.48) from control. However, carbenoxolone significantly decreased RV transverse CV without significantly changing longitudinal CV. As a result, RV CV anisotropy significantly increased (2.48 ± 0.38) compared to control Conclusion: Cell size does not fully explain interventricular conduction heterogeneities under control conditions. However, the preferential increase in RV anisotropy during gap junction uncoupling is consistent with larger RV cell geometry anisotropy. Lastly, conflicting experimental findings on the relationship between functional gap junction expression and conduction velocity may be in part dependent on the regions where the measurements were made.
2.20 A Novel Technique to Quantify Connexin Co-expression Ratios
Giselle Rowlinson1, Emmanuel Dupont1, Neil Thomas1, Piers E.F. Daubeney2, and Nicholas J. Severs1
1National Heart and Lung Institute, Imperial College London and 2Royal Brompton Hospital, London, UK
Cardiac myocytes express three connexins, Cx43, Cx40 and Cx45. We have developed a western blot technique that enables the quantification of the relative expression levels of these three connexins. This technique can be applied to human cardiac tissue to define connexin expression profiles of different regions of the normal heart, and alterations in these profiles in disease. It also has applications in cell lines such as the differentiated mouse atrial myocyte, HL-1, where accurate determination of connexin co-expression stoichiometry is necessary to relate the structure of connexons to their function. The technique uses HeLa cell lines transfected either with native Cx43, Cx40 or Cx45, or with a V5 “tagged” version of these connexins. The V5 tag provides a common epitope that permits the use of the same antibody to detect different molecules. The amounts of tagged connexins are standardised using the V5 signal intensity and thereafter the tagged and untagged connexins are detected together using specific anti-connexin antibodies. This allows us to calculate the levels of native Cx43, Cx40 and Cx45 in these transfectants. The HeLa cell lysates are then diluted such that each sample contains an identical number of connexin molecules per unit volume. These standardised lysates are loaded alongside the test samples for detection and quantification. Apart from the quantification of relative connexin co-expression, the loading of standardised amounts of connexins permits comparison between membranes blotted at different times. We have now calculated the relative quantities of Cx40 and Cx43 in human paediatric atrial tissue (Cx40: 51.4 ± 3.4%, Cx43: 48.6 ± 3.4%, Mean ± SD). This technique applied to the differentiated HL-1 cell clones or to inducibly transfected cell lines allows relative connexin quantities to be correlated with the conduction velocities of the different cell lines. (See abstract Dias et al.)
2.21 Signal Transduction of the Adrenergic Control of the Cardiac Connexin43 Expression
Aida Salameh1, Silke Holtz2, Jan Schulte3, Dietrich Pfeiffer2, Jan Janousek4, and Stefan Dhein3
1Clinic for Pediatric Cardiology, University of Leipzig; 2Medizinische Klinik I, Abt. Kardiologie, Leipzig; 3Herzzentrum, Klinik für Herzchirurgie, Leipzig; and 4Klinik für Kinderkardiologie, Herzzentrum Leipzig
In the heart gap junctional communication forms the basis for a regular rhythmic beating and regular electrical propagation. The most prominent cardiac gap junction protein is connexin43 (Cx43). Increased Cx43-expression has been identified in cardiac hypertrophy and may lead to arrhythmia. Besides acute effects on gap junction channel function, chronic regulation of Cx43 expression can affect intercellular communication. Since catecholamines play an important role in cardiac physiology and pathophysiology, we wanted to elucidate whether sub-chronic (24 h) alpha-adrenoceptor stimulation with phenylephrine (Phe) or beta-adrenoceptor stimulation with isoproterenol (Iso) may affect Cx43-expression. Cultured rat neonatal cardiomyocytes were exposed to either 0,0001-1 μ M Phe or 0,0001–1 μ M Iso for 24 hours. First, Cx43-expression was determined by Western blotting and PCR. Both Phe and Iso lead to a significant increase in Cx43 protein and mRNA expression with logEC50 of −8.2+/−0.01 and logEC50 -8.14+/−0.05, respectively. This increase in Cx43-expression was accompanied by a significant increase in gap junction current assessed by dual whole cell voltage clamp. The effect of Phe could be inhibited by prazosin and bisindolylmaleimide I (PKC-inhibitor), while the Iso effect was antagonised by propranolol and H8 (PKA-inhibitor), but not vice versa. To elucidate the further signal transduction cascade we investigated phosphorylation of ERK1/2, p38 MAPK, JunK and Akt. To our surprise both, alpha-and beta-adrenergic stimulation led to phosphorylation of ERK1/2, JunK and Akt, while p38MAPK was only very slightly affected, indicating convergence of both pathways at the final regulation of Cx43-expression. Finally, we investigated nuclear translocation of transciption factors by EMSA and Western blot, detecting enhanced AP1 presence (144 ± 4% (Iso), 149 ± 6% (Phe)), and CREB (135 ± 4 (Iso), 128 ± 2 (Phe)), also indicating convergence of the pathways. Thus, adrenergic stimulation affects cardiac gap junction expression via MAPK system and –as Akt is concerned-phosphatidylinositol signalling, and may account for the changes seen in cardiac hypertrophy.
2.22 Spatial Association of Cxs and Sites of Ca2+ Modulation in Intact Resistance Arteries: Functional Implications of Myoendothelial Microdomain Signaling?
Shaun Sandow
Pharmacology, School of Medical Sciences, University of New South Wales, Australia
Current and/or small molecule transfer via gap junctions (GJs) coupling vascular cells is integral for coordinated dilator and constrictor function, and thus for vascular tone. Vasodilator function is associated with heterocellular GJs between endothelial cells (ECs) and smooth muscle cells (SMCs), as myoendothelial (ME) GJs, and with homocellular EC and SMC GJs. Modulation of Ca2+ is critical for vascular function and the spatial association of GJ connexins (Cxs) and sites of Ca2+ release and action are likely functionally linked. In rat mesenteric arteries, adjacent ECs are coupled by Cx37, 40 and 43 GJs, which are associated with densities of small conductance Ca2+-activated potassium channels (SKCa); whilst Cx37 and 40 MEGJs are associated with densities of intermediate (I) KCa. The aim of this study was to further determine the high resolution spatial association of Cxs and selected Ca2+ modulation sites that are integral for vascular function. Rat mesenteric artery segments were high pressure frozen, freeze-substituted and lowtemperature embedded. Serial consecutive sections were incubated with multiple Abs to IP3R, TRPC3, TRPV4 and Cxs37, 40, Cx43 and secondary 5 and 10 nm Au conjugate. Confocal labeling of fixed flat tissue segments was also carried out using the same Abs and secondary Alexa conjugates. Confocal staining was overlayed with IEL autofluorescence to show the relationship between Cxs, IP3R, TRP and IEL holes (as MEGJ sites). Intense IP3R was present at the intimal MEGJ site. TRPC3 was present at the MEGJ surface membrane, whilst TRPV4 was diffusely localized to the EC membrane; with some localized cytoplasmic staining. The differential spatial localization of Ca2+ modulation sites and vascular Cxs suggests a causal relationship of their functional activation in that these sites of current transfer and Ca2+ modulation may interact. Such interactions may represent a selective target for the control of vasodilator function and vascular tone.
2.23 Protective Role of Endothelial Cx40 in Lung Inflammation
Klaus Ernst Ludwig Scheckenbach1, Antony AB Van Veen2, Maya Richani Sarieddine3, Habo J Jongsma2, Brenda R Kwak4, and Marc Chanson3
1HUG, Geneva, Switzerland; 2Department of Medical Physiology, Utrecht University, the Netherlands; 3Department of Pediatrics, HUG, Geneva, Switzerland; and 4Division of Cardiology, HUG, Geneva, Switzerland
The expression of Cx43 and its contribution to the propagation of pro-inflammatory signals has been reported in endothelial cells of the lung capillary bed. Whether Cx40 and Cx37, two other endothelial connexins are also involved is not known. To address this question, we studied lung inflammation evoked by intratracheal instillation (IT) of Pseudomonas aeruginosa endotoxin (LPS) in mice with endothelial-specific deletion of the Cx40 gene (Tie2Cre+Cx40fl/fl) using the Cre-LoxP system. Cx40 was immuno-localized in the endothelium of small vessels and in alveolar septa of control (Tie2Cre-Cx40fl/fl and Tie2Cre+Cx40wt/wt) mice but not of Tie2Cre+Cx40fl/fl animals. Cx37 was not immuno-detected in alveolar septa and endothelial specific deletion of Cx40 did not affect the expression level of Cx37 and Cx43 in total protein lung extracts. In control mice, LPS evoked an inflammatory response characterized by the rapid recruitment of neutrophils into the airways, as evaluated by scoring leukocytes collected by bronchoalveolar lavage (BAL) 3 and 6 hours after IT. LPS IT did not change the level of Cx40 expression in these control mice. Interestingly, the number of neutrophils recovered from BALs of Tie2Cre+Cx40fl/fl mice was markedly increased as compared to control animals. This enhanced recruitment of neutrophils in Tie2Cre+Cx40fl/fl mouse airways was caused by differential priming of the leukocytes. Indeed, in vitro assays demonstrated the increased adhesion to and transmigration across an endothelial cell monolayer of neutrophils isolated from Tie2Cre+Cx40fl/fl mice as compared to control leukocytes. This differential priming of neutrophils was strengthened by the presence of larger amount of acute-phase cytokines, including TNF-alpha, INF-gamma, IL-6 and IL-12, in BALs of Tie2Cre+Cx40fl/fl mice. These results suggest that Cx40 in the lung capillary bed contributes to early anti-inflammatory mechanisms that limit neutrophil emigration to the airways. Thus, endothelial Cx40 may play a protective role in the pathogenesis of lung inflammation.
2.24 Inhibition of Gap Junctional Coupling by Nicotine in Endothelial Cells is not Mediated by the A7 Subunit of the Nicotinic Acetylcholine Receptor
Anja Scheffler, Stefan Haussig, Nicole Dürrschmidt, Friedrich Wilhelm Mohr, and Stefan Dhein
Heart Centre Leipzig
Introduction: Smoking is still one of the most important causes of morbidity and mortality in industrialized countries particularly with regard to cardiovascular diseases. Therefore we investigated the effects of nicotine on endothelial cells and their intercellular coupling via gap junctions. Methods: Human umbilical vein endothelial cells (HUVEC) were cultivated for 5 days and then cells were washed, and gap junctional coupling was studied by dye transfer. Nicotine effects were tested at a concentration of 1 μ M, which corresponds to peak concentrations during cigarette smoking. To examine whether the effects were mediated through the α 7subunit of the nicotinic acetylcholine receptor we used the specific antagonist α -Bungarotoxin (concentration 10 nM). We also performed Western Blot of Connexin Cx 37, 40 and 43 as well as immunohistology. Results: After carefully washing out all nicotine, gap junctional coupling was decreased in cells grown under 1 μ mol nicotine (coupled cells after 5 minutes: 18.15 ± 0.80 (control) vs. 15.0 ± 0.76 (nicotine); n = 27; p < 0.05). This effect was not diminished by inhibition of the α 7subunit by α -Bungarotoxin (13.53 ± 0.95 in nicotine group vs. 13.18 ± 0.81 in nicotine plus α -Bungarotoxin group; n = 17; p = 0.78). α -Bungarotoxin itself also did not have any effect (16.82 ± 1.04 (n = 17) in control group vs. 16.93 ± 1.29 (n = 15) in α -Bungarotoxin group; p = 0.95). Western Blot and immunohistology have shown a down regulation in connexin Cx43 and Cx37 in cells grown under nicotine. Conclusions: From our results we conclude that nicotine leads to an inhibition of intercellular coupling via gap junctions in HUVEC. This effect does not seem to be mediated by the α 7subunit of the nicotinic acetylcholine receptor. Another subtype must be responsible for the effect. Our results may help to understand how smoking contributes to and worsens cardiovascular diseases.
2.25 Is Cx43 Involved in Antifibrillating Effects of PUFA and Atorvastatin in Rats Suffering from Dyslipidemia?
Narcisa Tribulova1, Vladimir Knezl2, and Marcela Fialova1
1Institute for Heart Research, Bratislava, Slovakia and 2Institute of Experimental Pharmacology, Bratislava, Slovakia
Our pilot study showed that rat model of hereditary hypertriglycerolemia (HTG) is accompanied by ventricular gap junction Cx43 remodelling that likely is implicated in increased susceptibility of those rats to ventricular fibrillation (VF). N-3 polyunsaturated fatty acids (PUFA) and some lipid-lowering drugswere reported to exhibit antiarhythmic actions, however, mechanisms involved are not clear. Therefore, we examined effects of PUFA and atorvastatin (ATO) on VF threshold, ultrastructure of the cardiomyocytes and their junctions as well as on Cx43 expression in HTG rat hearts. Experimentswere conducted on HTG and Wistar male rats fed with PUFA (40 mg/100 g BW) or ATO (0.05 mg/100 g BW) for 2 month and untreated controls. Isolated perfused heart model was used to test threshold for sustained VF. Ventricular tissues were taken for ultrastructure and Cx43 analysis. Results showed that VF threshold of untreated HTG rat heartswas significantly lower than control Wistar rats (15+1.2 mA vs 25+1.8 mA). In contrast, treatment of HTG rats with PUFA or ATO resulted in an increase of VF threshold to 40+2.1 mA and 45+1.8 mA (p < 0.05), respectively. Moreover, in three of eight PUFA-fed HTG rats sustained VF could not be induced even by repetitive 45 mA test stimulus. There were no apparent myocardial differences in distribution or amount of Cx43-immunopositive gap junctions between treated and untreated HTG rats and internalization as well as lateralization of gap junctions persisted upon treatment. However, subcellular examination revealed better preservation of cardiomyocyte structure including cell membrane and intercellular junctions integrity in both PUFA and ATO treated HTG rats. It is concluded that PUFA and ATO exert clear anti-fibrillating effects in rats suffering of dyslipidemia despite the arrhythmogenic gap junction Cx43 remodelling was not eliminated. Nevertheless, further studies are needed to examine as whether phosphorylation of Cx43 and channels function was affected due to PUFA or ATO treatment.
2.26 Functional Differences Between Atrial and Ventricular Gap Junctions as a Result of Connexin40 Co-Expression
Richard Veenstra1, Xianming Lin1, Joanna Gemel2, and Eric Beyer2
1SUNY Upstate Medical University and 2The University of Chicago
Atrial and ventricular gap junctions differ in their connexin expression, but the functional consequences of Cx40 co-expression with Cx43 in atrial myocytes has not been determined. We applied the cardiac action potential voltage clamp and unilateral spermine block protocols to cultured neonatal mouse atrial and ventricular myocytes to assess the transjunctional voltage (Vj)-dependent gating kinetics and overall contribution of Cx40 to atrial gap junction conductance (gj). Abundant Cx40 and Cx43 was detected in immunohistochemical fluorescent micrographs in cultured atrial myocytes. Cx43 was more abundant in quantitative western immunoblots of Cx40 and Cx43 protein, but two photon confocal imaging revealed Cx40 plaques sequestered in the center of the gap junction plaque labeled by Cx43 immunofluorescent localization. Spermine, which blocks only Cx40-containing gap junctions, reduced atrial gj by 52 ± 12% compared to 19 ± 9% in ventricular cell pairs when applied intracellularly in 2 mM concentrations to one side of the junction. Transjunctional voltage (Vj)-dependent inactivation during the cardiac action potential was reduced in atrial relative to ventricular gap junctions. The inactivation kinetics showed a greater contribution from a slow component observed in both Cx40 and Cx43 gap junctions and proportionately less of a 10-fold faster inactivation component associated with Cx43-expressing ventricular myocytes. Maximum gj inactivation during the action potential was 40% of peak gj for atrial and 60% for ventricular myocytes. During repolarization, the slope conductance was increased (“faciliation”) in both cell types, but the magnitude of the increase was greater in the ventricle. We conclude that the co-expression of Cx40 in atrial gap junctions alters the Vj-dependent gating that occurs with conduction delays > 1 ms/cell and introduces additional regulatory properties to cardiac gap junctions that may play a role in slow myocardial conduction and arrythmogenesis.
2.27 Spreading of Connexin43 into the Extrajunctional Plasma Membrane of Ischemic-Preconditioned Myocardium in the Rat
Friedrich Vetterlein1, Christian Mühlfeld2, Rolf Volkmann1, Cenc Cetegen1, and Gerhard Hellige1
1University of Göttingen, Abt. Anaesthesiol. Forschung and 2Anatomisches Institut Universität Bern
Spreading of connexin43 (Cx43) from gap junctions into the extrajunctional plasma membrane has been observed to develop during myocardial ischemia and to become accelerated by ischemic preconditioning (IP). Since it has been observed that connexin channels open when dephosphorylated, the question arose which changes in phosphorylation occur in extrajunctional Cx43, especially under IP. Therefore we studied regional myocardial ischemia in anesthetized rats and elicited IP by three short cycles of ischemia/reperfusion, followed by a period of ischemia of 15 or 45 min duration. Hearts were frozen and fluorochrome-immunostained for phosphorylated and dephosphorylated Cx43. Rela-tive fluorescence units (RFU) were determined from the ratios of light intensity in gap junctions and extrajunctional membranes, respectively, vs. that of myocyte interiors. In experiments without IP labeling of phosphorylated Cx43 revealed a decreasing gap-junctional fluorescence with prolongation of ischemia: 1.65 ± 0.27 and 1.29 ± 0.08 RFU (15 min and 45 min, respectively; non-ischemic zones: 1.69 ± 0.19 RFU). The data of the corresponding IP groups were 1.52 ± 0.08 and 1.19 ± 0.04 RFU. No immunofluorescence of phosphorylated Cx43 became discernable in the extrajunctional section of cell membranes. Staining of dephosphorylated Cx43 was found increased with prolonged ischemia. Labeling intensities in gap junctions were 1.80 ± 0.09 and 3.12 ± 0.55 RFU (15 min and 45 min ischemia, respectively; non-ischemic zones: 1.23 ± 0.06 RFU). The data of the corresponding IP-groups were: 2.10 ± 0.24 and 2.57 ± 0.33 RFU. In ischemic myocardium intense immunofluorescence of dephosphorylated Cx43 occurred in the extrajunctional membrane which also increased with duration of ischemia and was not significantly changed by IP (without IP: 1.06 ± 0.03 and 1.30 ± 0.07 RFU; with IP: 1.15 ± 0.05 and 1.27 ± 0.07 RFU; non-ischemic zones: 1.02 ± 0.01 RFU). In conclusion, IP did not prevent ischemia-induced dephosphorylation of Cx43 which is thought to favor unspecific cellular permeabilization. The assumption of changes in non-channel functions of Cx43 are more likely to explain IP-induced myocardial protection.
2.13 Endothelial Gap Junctions Are Down-Regulated by Arsenic Trioxide
Hung-I Yeh, Yusan Chou, and Cheng-Ho Tsai
Mackay Memorial Hospital, Taipei, Taiwan
We investigated the effect of As2O3, an anti-cancer drug, on endothelial gap junctions. Human aortic endothelial cells (HAEC) were treated with As2O3 at 1, 10, 100, and 1000 ng/ml and the cells were examined to evaluate the expression of connexin43 (Cx43) and gap-junctional communication function. Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) were measured to detect the presence of endothelial dysfunction. In addition, male Sprague-Dawley rats were given intravenous As2O3 (200 μg/kg/day) or saline for 4 weeks, and the endothelial gap junctions and eNOS as well as the circulating NO levels were evaluated. The results showed that in HAEC Cx43 gap junctions were down-regulated and the gap-junctional communication was attenuated by As2O3. Such a down-regulation of Cx43 gap junctions disappeared in the presence of the protease inhibitors leupeptin plus N-acetyl-Leu-Leu-Norleu-al and was associated with reduction of Cx43 transcripts. At the dose of 100 ng/ml, eNOS was reduced by 48 hours, however, NO was markedly reduced in 1 hour. In animals treated with As2O3, endothelial gap junctions made of Cx37, Cx40, or Cx43 were all down-regulated, while the eNOS and circulating NO levels remained stationary. In conclusion, in both the in vitro and animal experiments, endothelial gap junctions were consistently down-regulated by As2O3, unlike the responses of eNOS and NO, which differed according to the experiments. The reduction of Cx43 involved regulation at the transcription level and the degradation pathways. These findings suggest that gap-juctional communication in the vascular endothelium is inhibited in patients treated with As2O3.
Poster Session 2.2: Connexins in Development, Reproduction and Organogenesis
2.32 Opposing Effects of CNTF and Retinoic Acid on Connexin43 Gap Junctions During Differentiation of P19 and Olfactory Epithelial Cell Lines
Vania Figueroa1, Varvara Zeldovich2, Carla Pacheco3, Lorena Sülz3, Carolina Gatica4, Juan Carlos Sáez4, and Agustín D. Martínez2
1Universidad de valparaíso; 2Departamento de Neurociencias, Universidad de Valparaíso, Valparaíso, Chile; 3Facultad de Ciencias, Universidad de Chile; and 4Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile
Connexin43 (Cx43) is a gap junction protein highly regulated during brain development. Cx43 may influence neuronal differentiation by changing critical cellular features like gap junction channel permeability. To address this hypothesis we studied in vitro neurogenesis in P19 cells (mouse embryonic carcinoma pluripotent cell line) and OLF442 (olfactory epithelial cell line). Immunoblots and indirect immunofluorescence analysis showed that both cell lines express Cx43 and are coupled (diffusion of microinjected-Lucifer yellow to neighbouring cells) indicating the presence of functional gap junction channels. Retinoic acid (RA) plus cellular aggregation induced neurosphere formation and high levels of neuronal differentiation in P19 and OLF442 cultures (neuronal marker expression: alpha-III tubulin, GAP-43, neurofilament and NeuN). In addition, a progressive and concomitant reduction in both the total levels of Cx43 and dye coupling incidence were observed in the cultures during and after RA treatment. Serum-free culture conditions induced differentiation in OLF442 cells and reduction of Cx43 levels. On the contrary, treatment of P19 cells with ciliary neurotrophic factor (CNTF) and cellular aggregation induced a progressive increase in Cx43 and differentiation, but failed to produce mature neurons. However, increased differentiation was observed in cell cultures treated simultaneously with RA and CNTF. The effect was associated with changes in gap junction channel permeability. Finally, neurogenesis was reduced in P19 cells stably transfected with Cx43, suggesting an inverse relationship between Cx43 expression and neurogenesis. (Supported by grant: FONDECYT 1050857, DIPUV-33, ANILLO ACT-46).
2.34 Expression of Connexin32 and Connexin43, but Not Connexin26 in Human Normal Oesophageal Epithelia Is Under Direct Control of P63
Vladimir Krutovskikh and Hainaut Pierre
International Agency for Research on Cancer
The p63 protein has fundamental morphogenic roles in squamous epithelia, which in turn are characterized by specific cell adhesion properties. In normal oesophageal epithelia p63 is expressed at high levels in non-diffentiated, highly proliferating cells. With the help of the laser scanning confocal microscope, we found that the p63-positive cell population expresses a prominently different set of cell adhesion proteins compared to differentiated, p63 negative oesophageal cells. In particular, we found that three gap junction proteins—connexin26, connexin32 and connexin43 are overexpressed in p63 positive cells, but all these connexins were exclusively located in the cytoplasm. Inhibition of p63 expression by siRNA in human oesophageal squamous cell carcinoma TE cells in culture demonstrated that connexin32 and connexin43, but not connexin26, as well as several desmosomal, adherence and tight junctional proteins are regulated by the p63 protein. Furthermore, immunostaining showed that human squamous cell carcinoma of oesophagus are overexpressing connexin26, connexin32 and connexin43, which are retained intracytoplasmically. These observations further support the notion that p63 is a key regulator of squamous cell adhesion, and that its deregulation may have oncogenic effect in squamous cell carcinoma.
2.35 Different Cholesterol Distributions and Removal of Cholesterol in Gap Junctions Are Associated with Fiber Cell Differentiation in the Chicken Lens
Woo-Kuen Lo and Sondip K. Biswas
Departments of Anatomy and Neurobiology, Morehouse School of Medicine
Objectives: To determine the possible changes in the distribution of cholesterol in gap junction plaques during fiber cell differentiation and maturation in the chicken lens. The mechanism by which cholesterol is removed from gap junction plaques is also investigated. Methods: Visualization of cholesterol and endocytosis of cholesterol-rich vesicles associated with gap junction plaques were examined with filipin cytochemistry, freeze-fracture TEM and thin-section TEM. The nature of cholesterol-containing gap junctions was confirmed by combining filipin cytochemistry with freeze-fracture immunogold labeling (FRIL). Results: Chicken lens fibers contain cholesterol-rich, cholesterol-intermediate and cholesterol-free gap junction populations in both outer and inner cortical regions. Filipin cytochemistry and FRIL confirmed that Cx56 and Cx45.6 antibodies were labeled in the cholesterol-containing gap junctions. Quantitative analysis showed that approximately 86% of gap junctions in the outer young cortical zone were cholesterol-rich gap junctions, whereas approximately 81% of gap junctions in the inner mature cortical zone were cholesterol-free gap junctions. A number of pleiomorphic cholesterol-rich vesicles of varying sizes were frequently observed in the gap junction plaques. The internalization of these vesicles indicates their involvement in the removal of cholesterol from gap junctions through endocytosis. In addition, the connexons in all cholesterol-free gap junctions displayed a tight, hexagonal (crystalline) arrangement, whereas those of cholesterol-rich gap junctions were in a loose, random configuration in the adult lens, suggesting the role of cholesterol in regulating the connexon packing. Conclusions: Gap junctions in the outer young fibers are enriched with cholesterol because they are assembled in the cholesterol-rich cell membranes in the lens. A majority of cholesterol-rich gap junctions in the outer young fibers are transformed into cholesterol-free ones in the inner mature fibers during fiber cell maturation. A distinct endocytotic process of cholesterol-rich vesicles plays a role in the transformation of cholesterol-rich gap junctions into cholesterol-free ones during fiber cell maturation.
2.36 Cytoplasmic Connexin32 Protein Enhances Motility and Metastatic Ability of Human Hepatoma Cells in a Gap Junction-Independent Manner
Yasufumi Omori, Qingchang Li, Yuji Nishikawa, Toshiaki Yoshioka, Masayuki Yoshida, and Katsuhiko Enomoto
Department of Pathology and Immunology, Akita University School of Medicine
Connexins have long been believed to suppress tumor development during carcinogenesis by exerting gap junctional intercellular communication (GJIC). Although GJIC is abrogated in hepatocellular carcinoma (HCC), connexin32 (Cx32) protein tends to remain expressed in cytoplasm, but not in a cell cell contact area; thus, it is incapable of forming gap junctions. Hypothesizing that cytoplasmic Cx32 protein that has accumulated in HCC should have its proper functions, which are independent of GJIC, we established a Tet-off inducible expression system of Cx32 in human HuH7 and Li-7 HCC cells, both of which were unable to support the formation of Cx32-mediated gap junctions, so that Cx32 protein could be overexpressed by doxycycline (Dox) withdrawal. Although the established Tet-off Cx32 cells exhibited a 4-fold increase in Cx32 expression after Dox withdrawal, none of them were dye-coupled, and Cx32 protein was retained in the Golgi apparatus. However, the proliferation rate of the Tet-off Cx32 cells was significantly higher in the Dox-free medium than in the Dox-supplemented one. Transwell assays also revealed that Dox withdrawal enhanced serum-stimulated migration and invasiveness into the basement membrane of theTet-off Cx32 cells. Furthermore, when the Tet-off Cx32 cells were xenografted into the liver of SCID mice, only the mice to which no Dox was administered developed metastatic lesions, indicating that overexpression of cytoplasmic Cx32 protein induced metastasis of our HCC cells. Our results suggest that, while Cx32-mediated GJIC suppresses the development of HCCs, cytoplasmic Cx32 protein exerts effects favorable for HCC progression, such as invasion and metastasis, once the cells have acquired a malignant phenotype
2.39 Connexin Expression and Functional Analysis of Gap Junctional Communication in Mouse Embryonic Stem Cells
Philipp Wörsdörfer, Stephan Maxeiner, Christian Markopoulos, Stephanie Urschel, and Klaus Willecke
Universität Bonn, Institut für Genetik, Abt. Molekulargenetik, Bonn, Germany
Gap junctions are membrane conduits that allow direct intercellular diffusion of metabolites, ions and second messengers between contacting cells resulting in metabolic and electrical coupling. Gap junctional intercellular communication (GJIC) has been suggested to be involved in cell proliferation, differentiation and homeostasis. In this study, we have analyzed the expression of all mouse connexin mRNAs and proteins in mouse embryonic stem (ES) cells. Furthermore, we wanted to elucidate the function of gap junctional communication in cultured mouse embryonic stem cells, using pharmacological gap junction inhibitors. Transcripts of seven different connexin (Cx) isoforms were found by RT-PCR, while protein expression could only be demonstrated for connexin31, 43 and 45 by immunofluorescence, immunoblotting and the analysis of reporter gene expressing HM1 cells that were previously used to generate connexin deficient mice. Abundant GJIC monitored by dye-injection, could be abolished by pharmacological gap junction inhibitors (18-α -glycyrrhetinic acid, carbenoxolone), resulting in the initiation of apoptosis. This led to the hypothesis that intercellular communication might be necessary to maintain the survival of embryonic stem cells. In contrast, the decrease of gap junctional coupling to a similar extent as with inhibitors via downregulation of Cx31 and Cx45 expression by RNA interference in Cx43 deficient ES cells did not support this hypothesis. Thus we conclude that apoptotic cell death triggered by the pharmacological gap junction inhibitors is likely due to an unspecific side effect, beyond inhibition of GJIC. Co-immunoprecipitation experiments with purified hemichannels from HM1 ES-cells led to the assumption that Cx31 and Cx43 isoforms as well as Cx43 and Cx45 isoforms form heteromeric hemichannels while Cx31 and Cx45 do not. Results of immunofluorescence analyses demonstrate colocalization of all three ES cell connexins in the same gap junctional plaque.
WEDNESDAY, AUGUST 8, 2007 (15.10–17.30)
Poster Session 3
Posters 3.1–3.22
Poster Session 3.1: Connexins in Brain and Nervous Tissue
3.1 Lipid-Dependent Overexpression of the Icer-1 Repressor Decreases the Expression of Connexin36 in Insulin-Secreting Cells
Florent Allagnat1, Martin David1, Favre Dimitri2, Abderrahmani Amar2, Waeber Gérard1, and Haefliger Jacques-Antoine1
1Department of Medicine, University Hospital, CHUV, Lausanne, Switzerland and 2Department of Cellular Biology and Morphology, Lausanne, Switzerland
Metabolic syndrome is common in Western societies and includes dyslipidemia, high blood pressure and abnormal glucose homeostasis. Herein, we investigated the contribution of free fatty acids (FFA) and low density lipoproteins (LDL) to the expression and content of Connexin36 (Cx36), a gap junction protein known to be a critical contributor of glucose-stimulated insulin secretion. Chronic exposure to the saturated FFA palmitate and stearate reduced the expression of Cx36 in several insulin-secreting cell lines and freshly isolated mice islets whereas the mono or polyunsaturated FFA oleate or linoleate were ineffective. The pro-atherogenic modified oxidized LDL (oxLDL) particles also decreased Cx36 content whereas native LDL particles had no effect. These lipid-mediated effects were fully blocked by the protein kinase A (PKA) inhibitor H89, indicating that the cAMP/PKA pathway is involved in the Cx36 decrease. Transient transfection of a plasmid expressing the reporter gene luciferase under the control of a fragment of the Cx36 gene promoter revealed that palmitate and oxLDL decrease the Cx36 promoter activity. Mutation of a highly conserved cAMP response element (CRE) located in the Cx36 promoter resulted in abolition of the lipid effects. Palmitate and oxLDL increased the expression of the inducible cAMP early repressor (ICER-1), a known transcriptional repressor activated by PKA and binding to CREs. Overexpression of ICER-1 mimicked the effect of palmitate and oxLDL on Cx36 promoter activity, whereas ICER-1 repression prevented them. In conclusion, palmitate and oxLDL induce the expression of ICER-1, which subsequently inhibits the Cx36 gene expression. Considering the critical role of Cx36 expression levels on glucose-stimulated insulin secretion, the decreased Cx36 levels might contribute to the reduced glucose sensitivity observed during the pre-diabetic stage.
3.2 Pro-Inflammatory Cytokines Decrease the Expression of Connexin36 in Insulin-Secreting Cells
Florent Allagnat1, Klee Philippe2, Martin David1, Waeber Gérard1, Meda Paolo2, and Haefliger Jacques-Antoine1
1Department of Medicine, University Hospital, CHUV, Lausanne, Switzerland and 2Department of Cell Physiology and Metabolism, CMU, University of Geneva, Switzerland
Type I diabetes develops as a consequence of the autoimmune destruction of most of the insulin-producing α -cells composing the endocrine pancreas. Pro-inflammatory cytokines including the tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1α), and interferon gamma (IFNγ) are involved in α -cells apoptosis. We have shown that Connexin36 (Cx36) is a critical contributor of glucose-stimulated insulin secretion and protects α -cells from apoptosis. Herein, we investigated the contribution of cytokines to the expression of Cx36 in insulin-secreting cells. A 24 hour treatment with a combination of IL-1α, TNFα and IFNγ (Cytomix) strongly reduced the Cx36 mRNA and protein levels in insulin-secreting cell-lines and isolated mouse islets. To a lesser extend, IL-1α alone decreased the Cx36 levels in a dose-dependent manner, whereas TNFα and IFNγ had no effect. Metformin and AICAR, two compounds known to activate the AMP-dependent protein kinase (AMPK), reduced Cx36 mRNA and protein levels of insulin-secreting cells in a dose- and time-dependent manner, and their effects were blocked in presence of the AMPK inhibitor compound C. Interestingly, the Cytomix and IL-1α effects on Cx36 expression were also abolished following the AMPK inhibition, and the AMPK phosphorylation was strongly increased by the cytokines treatment. Strikingly, compound C prevented the cytokine-dependent α -cell apoptosis, suggesting that AMPK is involved in the cytotoxic effect of pro-inflammatory cytokines. Taken together, our results indicate an inverse relationship between the level of Cx36 expression and the degree of apoptosis, which is related to AMPK activity. Considering that Cx36 protects pancreatic α -cells against a variety of cytotoxic conditions, the data suggest that the AMPK-dependent decrease in Cx36 levels may contribute to the pathogenesis of type I diabetes.
3.3 Connexin32 Expression Determines NG2+ Progenitor Cell Fate in the Injured Hippocampus
Steffany Bennett1, L. Melanson-Drapeau1, M.Y. Morin1, S. Haykal1, S.N. Whitehead1, S. Boucher1, A.L.O. Hebb1, C. Messier2, and D.L. Paul3
1Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada; 2Department of Psychology, University of Ottawa, Ottawa, Ontario, Canada; and 3Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA
NG2+ progenitor cells are capable of neurogenesis in the uninjured adult hippocampus yet fail to participate in neuronal replacement following hippocampal damage. Here, we identify the gap junction protein connexin32 (Cx32) as a key intrinsic determinant of NG2+ cell fate. We found that Cx32 is expressed by hippocampal NG2+ cells activated by kainic acid-induced seizure. We showthat this expression is sufficient to inhibit neurogenesis in vivo and in vitro. Moreover, ectopic Cx32 expression in primary murine and human NT2/D1 precursor cells blocked neuronal differentiation in response to neurogenic stimuli. In loss of function studies, Cx32 deletion enabled NG2+ cells to generate new neurons following kainic acid-induced injury restoring behavioral indices of hippocampal-dependent learning and memory. Together, these data identify Cx32 as a possible therapeutic target that can be used to regulate NG2+ neuronal or glial replacement following brain injury. Supported by CIHR and OMHF to SALB and GM37751 to DLP; LMD and SB were supported by CGS and NSERC studentships.
3.4 Pattern of Expression of Connexin 43 in Human Gliomas: New Insights
Sophie Crespin1, Gaëlle Fromont2, Pierre Marie Levillain2, and Marc Mesnil1
1Université de Poitiers CNRS UMR and 2Service d'anatomie et cytologie pathologiques, CHU de Poitiers
Accumulating data have shown for decades a close association between cancer cell phenotype and disturbance of connexin expression. Several approaches attempting to modify the connexin expression tend to demonstrate that connexins are involved in cell growth regulation. However, most of these data were collected from in vitro and murine models. In order to understand better what is the relationship between connexin and human cancer process, we examine the intrinsic levels of expression and the localization of Cx43 in a panel of human gliomas (18 patients diagnosed with low grade and 40 with high grade). First, among 8 high-grade tumors studied by western blotting, half of them showed variable expression of Cx43 whereas for the same diagnosis others did not express the protein. An immunohistochemistry approach on all samples permitted to estimate the heterogeneity of tumors. Cx43 was shown at cell-cell contacts of GFAP-positive cells within peritumoral areas. The high heterogeneity of Cx43 expression was observed inside the tumor where the lack of detection, cytoplasmic localization and even a nuclear signal were present. The classic cytological and histological approaches failed to explain the difference in the pattern of expression of Cx43 between tumor cells within high-grade area. The use of specific cell markers seemed to indicate that the expression of Cx43 in the cytoplasm could be associated with GFAP-positive cells, whereas the nuclear signal was found in cells that did not express usual glial markers. Taken together, these data show that the level of Cx43 expression is not only inversely correlated with the grade of human gliomas; more than the level of expression, the cellular localization of the Cx43 should be taken into account and could play an important role in tumor process.
3.5 3D Modelling of Connexin Expression in Postnatal Neural Progenitor Cells
Stephen Fai1, Steffany A.L. Bennett2, and Lianne G. Gauvin2
1Architecture, Carleton University and 2Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemsitry, Microbiology, and Immunology, University of Ottawa
New neurons and glia are generated in adult brain by rare populations of activated neural stem and progenitor cells (NPCs). As a result of asymmetric division, these single cells can both self-renew and produce intermediate progeny that terminally specify to functional central nervous system cells. Determining how the fates of two initially identical daughter cells diverge within the same microenvironment is key to facilitating therapeutic cell replacement. Evidence that postnatal NPCs express a significant number of connexin proteins has raised new questions about the roll of cell-cell interaction in determining NPC fate. Our understanding of these interactions is hampered by the fact that most studies rely on 2D representations to describe the 3D microenvironment. Here, we employed an interdisciplinary approach to assess connexin expression in NPCs cultured as 3D neurospheres. Using multiple digital simulation technologies used by engineers, medical researchers, and architects, we show that specific connexins exhibit not only medial-lateral specificity (i.e. localize to cells in core or periphery of the neurosphere) but also a unique anteriorposterior polarity with respect to proximity to the airmedia interface. Localization of specific connexins within this spatial gradient to different NPC lineages suggests a structural determination of NPC fate that can be altered by connexin deletion. Data are presented as a series of quick-time movies describing these spatial relationships and their effect on NPC specification.
3.8 Connexin Expression by Postnatal Hippocampal Progenitor Cells is Dependent Upon Cell-Cell and Cell-Extracellular Matrix Interactions
Sophie Imbeault1, L.G. Gauvin1, A.C. McLean1, A.S. Menzies2, D.L. Paul2, A. Simon3, and S.A.L. Bennett1
1Neural Regeneration Laboratory, Department of Biochemistry, Microbiology and Immunology, University of Ottawa; 2Department of Neurobiology, Harvard Medical School; and 3Department of Physiology, University of Arizona
It has been clearly established that neural stem and progenitor cell populations (NPCs) in the subgranular zone of the dentate gyrus (SGZ) can generate new neurons and glia in adult brain. Accumulating evidence implicates connexin-mediated communication in the control of NPC survival and proliferation during CNS development but an influence of connexins on postnatal NPC fate has not been established. A prerequisite would be the expression of connexins by postnatal NPCs. To characterize this we performed RT-PCR, Western blotting and immunocytochemistry on neonatal NPCs isolated from the SGZ under a variety of culture conditions. We found that NPCs cultured in suspension as neurospheres, in the presence of EGF and bFGF, express Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein. Cx32 and Cx36 were detected at the mRNA level only. In cryostat sections of neurospheres, we localized Cx26, Cx30, Cx37, Cx40, and Cx45 to Type 1 or 2 nestin-positive NPCs while Cx29 was expressed by PDGFαR-positive glial progenitors. Negative controls included neurospheres cultured from null-mutant mice for each connexin except Cx26. Adhesion of NPCs to a laminin substrate altered their connexin protein profile inducing Cx36, inhibiting Cx47, and reducing Cx40 and Cx43 protein expression. Together these data demonstrate that proliferating postnatal NPCs express a significant number of connexins and that their expression patterns are influenced by cell-cell and cell-extracellular matrix contact. Supported by CIHR and OMHF to SALB and GM37751 to DLP; SI and LGG hold NSERC studentships.
3.9 Connexin35 Gap Junctions Exist in a Variety of Phosphorylation States in the Retina
Wade Kothmann, Xiaofan Li, and John O'Brien
University of Texas Health Science Center at Houston
Connexin35/36 (Cx35) is the most widespread neuronal gap junction (GJ) protein in the CNS. Electrical and tracer coupling are regulated by light adaptation in multiple retinal circuits that express this connexin. Cx35 has been shown to be regulated by PKA activity in retinal neurons and PKA phosphorylation in cell culture systems. This suggests that the phosphorylation state of Cx35/36 may report the coupling state between two cells. We examined the phosphorylation state of Cx35 in retina under different lighting conditions using phosphospecific antibodies against phosphoSer110-Cx35 and phosphoSer276-Cx35. Western blots of bass retinal membranes showed Cx35 to be phosphorylated at both Ser110 and Ser276 sites. This labeling was eliminated by alkaline phosphatase digestion. The homologous sites of mouse and rabbit Cx36 were also phosphorylated in retinal membranes. Bass retinas were collected either dark-adapted 2 hours before light onset or light-adapted 2 hours after light onset. Confocal immunofluorescence analysis of these retinas showed GJs identified with a monoclonal anti-Cx35 antibody to have variable phosphorylation at both Ser110 and Ser276 sites. Unusual GJs identified by their large size (4.9 to 32 μm2) and location in the inner plexiform layer showed a shift from heavily phosphorylated in nighttime, darkadapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites followed this pattern. Under both lighting conditions other GJs varied from non-phosphorylated to heavily phosphorylated. We conclude that retinal Cx35 exists in different phosphorylation states which are at least partially related to the population of GJs in which it resides. This state can change with lighting conditions. We predict that changes in the phosphorylation state of Cx35 reflects changes in coupling strength, and is likely controlled at the level of individual cell types or possibly individual GJs.
3.12 The Influence of Connexin 43 on the Proteome of Primary Mouse Astrocytes
Stephan Olk1, Andrey Turchinovich1, Michael Grzendowski2, Kai Stühler2, Georg Zoidl3, and Rolf Dermietzel3
1International Graduate School of Neuroscience, Ruhr-University Bochum; 2Medical Proteome Center, Ruhr-University Bochum; and 3Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum
Recent studies imply a role of the astrocytic gap junction protein connexin 43 (Cx43) in the regulation of important cellular processes including proliferation, migration and apoptosis. These mechanisms are considered to impinge on the proteome of the Cx43 expressing cells. To analyze the influence of Cx43 on the proteome we used RNA interference to downregulate the Cx43 expression in primary cultures of mouse astrocytes. We applied the Differential Gel Electrophoresis (DIGE) technique to compare astrocytes with downregulated Cx43 and control cells. The differential proteome analysis revealed eight significantly regulated proteins, which could basically be classified into cytoskeletal proteins and metabolic enzymes. Astrocytes treated with Cx43 siRNA showed a significantly increased expression of the cytoskeletal proteins beta-actin, GFAP and the tropomyosin alpha-4 chain. These results are in accordance with the putative roles of Cx43 in proliferation and migration, where a rearrangement of the cytoskeleton is essential. A future goal of our studies will focus on the analysis of the structural phenotype to specify the cytoskeletal changes. Among the regulated metabolic enzymes two proteins are of interest, which result in a decreased biosynthesis of cholesterol via the mevalonate pathway. Cx43 has been shown to target to cholesterol-rich lipid rafts. A current work is initiated to prove whether lipid rafts are influenced by siRNA mediated Cx43 downregulation. A further study centers on the analysis of the phosphoproteome. The rationale behind this study is to uncover a role of Cx43 in downstream signaling events, which include potential regulation of signaling pathways via protein kinases. Currently we are able to detect 300 to 400 phosphoproteins by applying two-dimensional Western blotting. We are confident to analyze changes of the phosphoproteome by exploiting that technique on Cx43 downregulated astrocytes. The next step will comprise the identification of effector proteins to unravel the yet uncharacterized signaling pathways affected by Cx43.
3.13 Connexin30 Is Implicated in Adult Neurogenesis
Hadi Daood Toeg1, Shawn N. Whitehead2, and Steffany A.L. Bennett2
1University of Ottawa (Canada), Faculty of Medicine, Biochemistry Department and 2University of Ottawa, Canada
The adult mammalian brain contains neural stem cells and neural progenitor cells (NPCs) with the capacity to proliferate and differentiate into functional neurons and glia. The majority of new neurons in adult brain derives from cells expressing nestin and glial fibrillary acidic protein (GFAP). These multipotential cells generate nestin+ intermediate progeny that retain some capacity for both neurogenesis and gliogenesis. The progeny of nestin+ cells commit to a neuronal lineage by specifying to doublecortin+ (DCX+) neuroblasts that then terminally differentiate to neurons. Cell-cell contact between “instructive” hippocampal astrocytes and multipotential NPCs has been shown to promote this transition to an immature neuronal phenotype. Underlying signaling mechanisms have only begun to be elucidated. Here, we show, for the first time, that Cx30 is expressed not only by hippocampal astrocytes but also by subsets of multipotential nestin+ NPCs in adult mice. Cx30 expression is lost once intermediate progeny irreversibly commit to a neuronal lineage as indicated by DCX expression. Using adult Cx30 null mutant mice, we show that Cx30 deletion does not impact on the overall rate of cell proliferation as assessed by bromodeoxyuridine labeling. Loss of Cx30, however, increases the percentage of actively proliferating nestin+ NPCs and decreases the percentage of actively proliferating DCX+ cells. Finally, we show that direct contact between astrocytes and postnatal NPCs is sufficient to direct nestin+ NPCs to differentiate to neurons when cultured in vitro. Together, these findings suggest that Cx30 mediated astrocyte-NPC communication may promote transition of a multipotential NPC towards a neuronal phenotype. Supported by CIHR and OMHF to SALB; HT holds a NSERC studentship.
3.15 The Polypyrimidine Tract Binding Protein (PTB) Interacts with the Internal Ribosome Entry Site of Zebrafish Connexin55.5
Georg Zoidl, Mahboob-ul-Hussain, and Rolf Dermietzel
Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, University Street 150, Bochum, Germany
We have identified an unusual internal ribosome entry site (IRES) present in the coding region of the zebrafish connexin55.5 (Cx55.5) that mediates the internal translation of a carboxy-terminal domain termed p11-CT. This domain is expressed in vivo in horizontal cells and in vitro in recombinant cells in addition to the full-length 55.5 kDa protein. In order to gain further insight into the mechanisms that promote the generation of this domain we characterized the IRES element in terms of the sequence elements necessary for its activity and the protein factor(s) which may play a role for its function. In silicio sequence analysis showed the presence of two stretches of polypyrimidine tracts designated PPT1 and PPT2 which influence the IRES activity in vitro. Selective deletion of PPT1 results in a decrease of the IRES activity, while the deletion of PPT2 results in a complete activity loss. RNA-EMSA and UV-cross linking experiments showed that a number of proteins form complexes able to bind to the IRES element. The first interacting protein characterized is the polypyrimidine tract binding protein (PTB). We could demonstrate that the PPT1 and PPT2 sites are required for the polypyrimidine tract binding protein (PTB) to bind, to influence IRES activity and hence formation of the p11-CT domain of Cx55.5. Our results demonstrate that PTB conveys a definite role in the regulation of the IRES activity of zfCx55.5. The possible functional implications on horizontal cell electrical coupling and a potential role during dark/light adaptation will be discussed. Supported by DFG and SFB 509 “Neurovision” (R.D.; G.Z.) and the International Graduate School for Neurosciences (IGSN, M.UH).
3.16 Generation of a Carboxy-Terminal Domain of Zebrafish Connexin Cx55.5 by Internal Translation Initiation In Vivo and In Vitro
Georg Zoidl1, Mahboob Ul-Hussain1, Jan Klooster2, Maarten Kamermans2, and Rolf Dermietzel1
1Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, University Street 150, D-44801 Bochum, Germany. 2Research Group Retinal Signal Processing, Netherlands Brain Research Institute, Amsterdam, The Netherlands
Changes of interneuronal electrical coupling in response to light adaptation and receptive field shapening is a key property in the horizontal cell/receptor cell (HC/RC) complex of the retina. It is anticipated that the regulation of these processes requires information transfer to the nucleus by locally generated messengers. Electrical synapses may comprise one pathway to drive such an signal. We have previously shown that the zebrafish Connexin55.5 (Cx55.5) and Cx52.6 are connexins with horizontal cell-restricted expression (Dermietzel et al., JN, 2000; Zoidl et al., JBC, 2004). In particular Cx55.5 forms hemichannels at dendritic sites where light-dependent plasticity occurs (Schields et al., JCN, 2007). Here we demonstrate the generation of a carboxy-terminal domain of Cx55.5 which may convey such a signaling function. This protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame start codon involving a putative internal ribosome entry site (IRES) localized in the coding region of Cx55.5. This protein product resembles an 11 kDa domain of Cx55.5 (p11-CT) and is partially located in the nucleus in vivo and in vitro. Changes in light conditions affect the formation of both Cx55.5 proteins in vivo. The nuclear occurrence of a fragment of an electrical synapse protein makes a putative cytoplasmic-nuclear signal transfer involved in gene regulation for structural plasticity at the HC/RC complex feasible. Supported by DFG and SFB 509 “Neurovision” (R.D.; G.Z.), the International Graduate School for Neurosciences (IGSN, M.UH) and KNAW (M.K.).
Poster Session 3.2: Channel Structure and Function
3.17 Gap-FRAP Technique Applied on Spheroid Three-Dimensional Model with the Confocal Scanning Laser Microscope
Muriel Abbaci1, Barberi-Heyob Muriel1, Blondel Walter1, Dumas Dominique2, Guillemin François1, and Didelon Jacques1
1Centre Alexis Vautrin-CRAN, UMR CNRS-UHP-INPL, Nancy-University; 2LEMTA, Equipe Mécanique et Ingénierie Cellulaire et Tissulaire-Service Imagerie Cellulaire; UMR CNRS-INPL-UHP, Nancy-University
Introduction: It is assumed that gap junctional intercellular communication (GJIC) is of great importance in the regulation of cell differentiation and cell growth. Numerous studies show that a majority of malignant cells are characterized by a decrease of number and functionality of gap junctions. Gap-Fluorescence Recovery After Photobleaching (FRAP) technique is adapted for quantifying modifications in functionality of GJIC in vitro. This paper presents a study of GJIC capacity on multicellular three-dimensional (3-D) model with the confocal scanning laser microscope using gap-FRAP technique. Materials and Methods: Intercellular communication of two human head and neck carcinoma cell lines (FaDu and KB cells) was performed in monolayer cultured cells and in 500 μ m diameter multicellular spheroids. Functionality of GJIC was compared with two fluorescent dyes, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). We recently demonstrated that calcein-AM was the most adapted tracer for 3-D approaches. Results and Discussion: In monolayer cultured cells, FaDu cell line only displayed Cx43 expression around nucleus associated with a weak fluorescent recovery. On the contrary, KB cells showed Cx43 expression at the cell-cell borders correlated with a fluorescence recovery 15-fold higher than for FaDu cells. The fluorescence recovery kinetics obtained were fitted using an single exponential function. In 3-D model, KB spheroids were characterized by a significant fluorescence recovery after photobleaching and FaDu spheroids by no fluorescence recovery. However, recovery percents in KB spheroids were low compared to those obtained in monolayer cultured cells. Moreover, the fluorescence recovery kinetics appeared bi-exponential. Independently of cell lines, photobleaching and fluorescence recovery gradients were observed from center to periphery of photobleaching area (50 μ m). In order to assess quantitative parameters characterizing spatial distribution of FRAP in 3-D, the development of adapted behavioural models appears an essential condition.
3.21 Cell-Cell Transfer of Cyclic Nucleotides: Quantification and Comparison of Connexin-Dependent Permeability
Giedrius Kanaporis1, L. Valiuniene2, G. Mese2, T.W. White2, P.R. Brink2, and V. Valiunas2
1Stony Brook University, Stony Brook, NY, USA and 2Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA
Gap junction channels exhibit connexin dependent biophysical properties, including their diverse selective intercellular passage of larger solutes, including second messengers. Here, we report the direct investigation of cyclic nucleotide (cAMP) and Lucifer Yellow (LY) permeability through gap junction channels composed of different connexins. We quantified and compared cAMP and LY permeability in HeLa cells transfected with Cx26, Cx40 and Cx43. The quantitative channel permeability was determined using simultaneous measurement of junctional conductance and intercellular transfer of LY and cAMP. For cAMP detection the recipient cells were transfected with a cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH and SpIH derived currents (Ih) were recorded from the cell expressing SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH transfected cell resulted in a 5-6 fold Ih current increase over time. cAMP transfer was observed for homotypic Cx43 channels over a range of conductance from 1nS to 27nS. However, cAMP transfer was detected only in well coupled cell pairs of Cx26 (> 15 nS) and Cx40 (> 20 nS). This analysis showed that Cx43 channels are ∼ 2–3 times and ∼ 4–5 times more permeable to cAMP than Cx26 and Cx40 channels, respectively. Previously, we reported that Cx43 channels were ∼ 5 times more permeable to LY than Cx40 channels and the LY/K+ permeability ratios were 0.0025 (Cx40) and 0.028 (Cx43). Here, we found that Cx26 channels were ∼ 3 times less permeable to LY than Cx43 channels and ∼ 1.8 more permeable than Cx40 channels, yielding the LY/K+ ratio of 0.0049. In conclusion, both LY and cAMP show a similar permeability order for the following connexins: Cx43 > Cx26 > Cx40, suggesting that these channels have distinct selectivity for larger solutes. Supported by AHA0335236N; NIH:GM55263, EY14604, DC06652, & EY13163
3.24 Cx36 Protects Pancreatic Beta Cells Against Cytotoxic Attacks
Paolo Meda1, Philippe Klee2, Florent Allagnat3, Manon Peyrou2, Anne Charollais2, Dorothee Caille2, and Jacques-Antoine Haefliger3
1University of Geneva; 2Department of Cell Physiology and Metabolism, University of Geneva, Switzerland; and 3Department Medicine, University of Lausanne, Switzerland
Type I diabetes develops when most of the pancreatic beta cells have been killed by an autoimmune attack, leaving a few survivors which can no more sustain an adequate insulin secretion. The mechanism whereby these cells survive for years to the immune attack in unknown. Using a variety of transgenic mice and in vitro models, we have investigated whether this mechanism implicates some connexin-dependent signaling. Here, we show that 1) loss of Cx36 sensitizes the beta cells of KO-Cx36 mice to a treatment with either streptozotocin or alloxan, 2 drugs which selectively kill beta cells; 2) over-expression of Cx36 protects the beta cells of transgenic RIP-Cx36 mice against the same drugs; 3) in vitro, islets isolated from the latter animals were also protected against a cocktail of cytokines (IL-1beta plus IFNgamma plus TNFalpha) present at the onset of diabetes; 4) islets isolated from KO-Cx36 mice were sensitized to the apoptosis induced by the same cytokines; 5) these different effects are accounted for by differences in the release of mitochondrial cytochrome C oxidase in the cytosol and in the ensuing activation of effector caspases, which were significantly lower in beta cells expressing Cx36, than in those lacking the connexin; 6) the Cx-dependent protection requires cell-to-cell contact, and can be also provided by inducing beta cells to express either Cx32 (RIP-Cx32 mice) or Cx43 (RIP-Cx43 mice), on top of the native Cx36. The data show that loss of Cx36 sensitizes pancreatic beta cells to a variety of cytotoxic conditions, suggesting that decreased Cx36 levels may contribute to the pathogenesis of type I diabetes, and that treatments increasing Cx36 may be of therapeutic value in both the prevention and treatment of the disease.
3.26 Spatiotemporal Inhibition of Gap-Junctional Communication by Multiphoton Excitation-Evoked Chromophore-Assisted Laser Inactivation (MP-CALI)
Masahito Oyamada, Takuji Tanabe, Yoshihisa Yamaoka, Ping Dai, Hideo Tanaka, and Tetsuro Takamatsu
Kyoto Prefectural University of Medicine
Genetic approaches to the connexins (Cxs) such as gene knockouts and RNA interference, which ultimately reduce Cx levels in the entire cell, have provided direct evidence that gap junctional intercellular communication is essential for tissue functions and organ development, and that its dysfunction causes diseases. However, it is possible that one kind of Cx performs different functions depending on its location and/or a particular stage of temporal events. Such spatiotemporal differences in protein function are difficult to analyze with conventional genetic approaches. In this study, we demonstrate by multiphoton excitation-evoked chromophore-assisted laser inactivation (MP-CALI), that specific gap junctions made of Cx-enhanced green fluorescent protein (EGFP) can be spatiotemporally inactivated by brief laser irradiation. Experiments were performed on a pair of HeLa cells connected via a small gap junction plaque (length, < 1 μ m) consisting of Cx43-EGFP. While junctional currents were measured using the dual whole-cell voltage-clamp method, a single point of the Cx43-EGFP plaque was irradiated with 850-nm femtosecond laser light with a laser irradiance of 2.7 MW/cm2 for 380 ms. We found that during the laser irradiation, the gap junction current decreased ?80%. However, the same irradiation did not change gap junctional currents between HeLa cell pairs expressing Cx43 tagged with monomeric red fluorescent protein (mRFP), indicating that MP-CALI at a wavelength of 850 nm specifically inactivates gap junctions made of Cx-EGFP. We also found that MP-CALI can inhibit dye coupling between specifically selected cell pairs. MP-CALI of Cx43-EGFP showed a laser power-dependent decrease of gap junction current, i.e., the threshold laser power for current reduction is between 1.0 and 1.3 MW/cm2. Reactive oxygen species (ROS) scavengers, edaravone and sodium azide, impaired the MP-CALI response, indicating involvement of ROS in MP-CALI. Thus, MP-CALI could be useful for the elucidation of new Cx functions.
3.27 Examination of Gap Junctional, Intercellular Communication by In Situ Electroporation
Leda Raptis1, Aikaterini Anagnostopoulou1, Jun Cao1, Adina Vultur1, and Kevin Firth2
1Queen's University and 2Ask Science products Inc.
We previously described a powerful technique where gap junctional, intercellular communication (GJIC) of adherent cells can be examined by in situ electroporation on a slide, part of which is coated with electrically conductive and transparent indium-tin oxide. An electric pulse is applied through an electrode placed on the cells in the presence of the tracking dye, Lucifer yellow (LY). The pulse causes LY's penetration into the cells growing on the conductive part of the slide, and the subsequent migration of the dye to the non-electroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination. This technique has been extensively exploited for the examination of GJIC of a large variety of adherent cell lines. However, this approach is not suitable for cells that do not adhere well, such as cells grown to high confluences, because the turbulence generated as the electrode is removed can cause cell detachment, which makes GJIC examination problematic. In this communication, we describe a slide configuration where junctional communication can be examined in the absence of an upper electrode: Cells are grown on two co-planar electrodes separated by a dam which diverts the electric field, rendering it vertical to the cell layer. The elimination of an upper electrode is especially valuable for the electroporation of sensitive cells, such as terminally differentiated adipocytes or highly transformed lines.
3.28 Neonatal Rat Cardiomyocytes Show Characteristics of Non-Homotypic Gap Junction Channels
Jan Sebastian Schulte1, Diana Rojas-Gomez2, F.W. Mohr2, and Stefan Dhein2
1Institute of Pharmacology and Toxicology, University of Münster and 2Clinic for Cardiac Surgery, University of Leipzig
Neonatal rat cardiomyocytes mainly coexpress the connexins Cx40, Cx43 and to a small amount Cx45 leading to the potential formation of mixed (heteromeric/heterotypic) gap junction channels. Using the double cell voltage clamp technique we investigated voltage dependent inactivation of gap junction channels between cell pairs of Cx40, Cx43 and Cx45 stably transfected HeLa cells and compared those data to data obtained from cell pairs of cultured neonatal rat cardiomyocytes isolated from 24h old Sprague Dowley rats. With two synchronized switch-clamp voltage-clamp amplifiers (SEC05 NPI Electronic) we applied long lasting pulses (2s) with transjunctional voltage gradients from -120 to +120 mV (10 mV steps) to the cell pairs. The measured instantaneous gap junction conductance (GJinst) decreased to a steady-state conductance (GJss) with the bell shaped ratio between the normalized conductance (GJss/GJinst) and the transjunctional voltage gradient being fitted to the two state Boltzmann curve: gss/ginst = (gmax-gmin)/(1 + exp[A(Vj-V0)]+gmin. In accordance to previously published data the Boltzmann curves were almost symmetrical for the Cx transfected HeLa cells indicating homotypic gap junction channels. The Vj values for the half-maximal inactivation differed between the single Cx-Types (Cx45 < Cx40 < Cx43) with GJmin values ranging between 0,2 and 0,5. Boltzmann curves fitted to data obtained from neonatal rat cardiomyocyte-pairs expressing both Cx40 and Cx43 showed an asymmetrical inactivation pattern and GJmin values higher than 0,5 which cannot be explained by the presence of pure populations of homotypic gap junction channels of either isoform. In conclusion we assume the additional presence of heterotypic and possibly even heteromeric gap junction channels in neonatal rat cardiomyocytes investigated in our laboratories.
3.29 The Carboxy Terminus of Connexin43 in Regulating Cell Growth and Motility
Wun Sin, Dave Bates, and Christian Naus
University of British Columbia
Connexins mediate signalings by permitting intercellular communication, but there is also evidence to suggest connexins may regulate growth via a junction-independent mechanism by binding to intracellular molecules with their C-terminal tail. Objective: To determine whether CCN3 mediates Cx43-dependent cell growth and motility. Methods: The knockdown of Cx43 or CCN3 was carried out in two tumor lines: C6 rat gliomas and human breast cancer cells Hs578T, using either siRNA duplexes or vector based shRNA. Standard cell growth analysis was used to determine growth characteristics. In vitro wound healing and transwell assays were used to evaluate cellular motility. Results: To deduce whether Cx43 mediates the observed differences in motility in subclones of C6 cells which endogenously expressed high (C6-H) or low (C6-L) levels of Cx43, we conducted assays on C6-H cells retrovirally infected with Cx43 shRNA. Coincident with the stable knockdown of endogenous Cx43, a decrease in motility was observed. As the presence of gap junction inhibitors did not reveal significant differences in cell motility, C6 cells transfected with full length or C-terminal truncated Cx43 (Cx43Δ CT) were subjected to motility assays to expose alternate mechanisms of Cx43-mediated motility. Cells expressing full length Cx43 exhibited increased motility while cells expressing Cx43Δ CT did not. CCN3 was recently identified as a Cx43 carboxy terminus tail associating protein. It belongs to a family of extracellular matrix associated proteins involved many cellular processes such as differentiation and migration. CCN3 is growth suppressive and overexpression of exogenous CCN3 reduced growth rate of breast cancer cells. A reduction of Cx43 in Hs578T breast cancer cells with Cx43 shRNA resulted in a decrease in CCN3 expression and an increased in growth rate. Conclusions: Our results suggest that the carboxy terminus plays an important role in Cx43-mediated glioma motility and that CCN3 is a strong candidate that mediates Cx43-dependent growth suppression.
3.30 Structural Characterization of the Connexin40 Carboxyl Terminal Domain and its Interaction with Other Connexin Intracellular Domains
Paul Sorgen, Denis Bouvier, Sylvie Chenavas, and Fabien Kieken
UNMC
Cx43, Cx40, and Cx45 co-expression in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle, leads to the formation of heteromeric/heterotypic gap junction channels. The physiological consequences of heterologous gap junction channel formation on intercellular communication remain unclear, however, functional studies of heteromeric connexons and channels demonstrate that these structures display biophysical properties that do not correspond to the homomeric forms. Because the sensitivity of different homomeric gap junctions to biochemical modulators varies widely, the question arises as to the types of interactions that occur between connexins to integrate regulatory signals in heteromeric channels. Evidence supports involvement of the carboxyl terminal (CT) domains in regulation of these heteromeric channels. Our goals are to use the CT and cytoplasmic loop (CL) domains from the connexin isoforms expressed mainly in the heart (Cx43, Cx40, and Cx45) to characterize pH-dependent hetero-CT dimerization and binding to CL domains. We have assigned all of the resonance peaks for the Cx40CT domain and initial structural studies indicate the presence of short helical domains similar to the Cx43CT structure. Additional NMR, yeast twohybrid, and FTIR studies suggest the Cx40CT homooligomerizes, as well as, forms a complex with the Cx43CT. Initial studies of the CL domain have been focused on solving the Cx43CL structure in order to define the Cx43CL residues involved in binding the CT domains. In general, these studies are expected to provide evidence that the types of interactions between the cytoplasmic domains of different connexin isoforms regulate heteromeric channels.
3.31 Comparative Mapping of Pore Lining Residues in Cx50 and Cx32 Gap Junction Channels Reveals Differences in Pore Topology
Masoud Toloue1, Derek Beahm2, and Bruce Nicholson1
1University of Texas Health Science Center San Antonio and 2Physiology and Biophysics Departments, State University of New York at Buffalo
Evidence has shown that gap junction channels discriminate between permeants according to size and metabolite type. Understanding of the molecular basis of this selectivity has been hampered by a lack of information on comparative pore structures of different connexins. Using the substituted cysteine accessibility method (SCAM), we have previously mapped the pore lining residues of open Cx32 gap junction channels with a biotinylated maleimide reagent (MBB) [Skerrett et al, (2002)]. Reactivity was identified along one face of M3, and in the cytoplasmic half of M2. Reactivity in M1 was difficult to interpret, as cysteine substitution at many of these sites significantly modified channel gating, suggesting that the mutated protein was not in a wild type conformation. We have now completed a comparison with a second connexin, Cx50, with a much larger single channel conductance than Cx32 (220pS, compared to 55pS), yet a significantly lower size cut-off for dyes (∼600 compared to > 760MW). Cysteine substitutions in M1 of Cx50, unlike Cx32, induced no phenotypic gating changes, but also showed no reactivity, indicating that M1 contributes minimally to the pore lining of open gap junction channels. In contrast, six sites in M3 of Cx50 were reactive, with 5 corresponding to sites mapped in Cx32, and one towards the extracellular end of the pore being shifted by one residue from the pattern in Cx32. Four sites were also reactive on one face of the cytoplasmic end of the M2 helix in Cx50, but of these, only one corresponded to a site mapped in Cx32. The patterns of reactivity indicate that the M2 helix may line the pore at a different angle in the two connexins, consistent with the hypothesis, based on comparative permeability studies, that gap junction pores of different connexins may differ significantly in their topology.
3.32 Truncation of the Cx43 CT Domain Reduces Ventricular Gap Junction Activation and Facilitation During the Action Potential
Richard Veenstra, Xianming Lin, Karen Maass, and Mario Delmar
SUNY Upstate Medical University
Truncation of the cytoplasmic carboxyl terminal (CT) domain of connexin43 (Cx43) slows the rate of transjunctional voltage (Vj)-dependent inactivation. To investigate whether Cx43 truncation at the position of lysine258 (Cx43K258stop or M257) alters the phasic changes in cardiac action potential gap junction conductance (gj), neonatal ventricular myocytes from homozygous M257 mice were dissociated and studied using the dual whole cell action potential voltage clamp technique. The inactivation kinetics were slowed and the maximal reduction in gj achieved during the action potential plateau was reduced from 54% in wild-type ventricular gap junctions to 20%. The Vj-dependence of the inactivation rate constants is still being determined. The normalized steady state junctional conductance—voltage (Gj − Vj) curve exhibited an increase in the minimal conductance value (Gmin) from 0.27 to 0.43 and a decrease in slope corresponding to a reduction in the gating charge valence from 2.7 to 2.0 respectively for wild-type and M257 ventricular gap junctions. More importantly, the slope of the M257 ventricular Gj—Vj curve during the return phase of the ± 120 mV, 200 ms/mV Vj ramp was lower than the inactivation curve, which is opposite of what is observed in wild-type ventricular gap junctions. Thus, the “facilitation” of Gj observed during the Vj recovery phase (e.g. action potential repolarization) in normal ventricle is associated with the presence of a functional Cx43 CT domain. We hypothesize that Gj inactivation during action potential propagation enhances conduction slowing that can develop into unidirectional or complete conduction block and that facilitation may promote arrhythmogenesis caused by reentrant excitation or focal triggered activity. Slowing ventricular gap junction inactivation and eliminating facilitation may protect against cardiac arrhythmias, but may also reduce the amount of protective uncoupling during prolonged ischemic events prior to infarction or sudden cardiac death.
3.33 Temperature-Dependent Modulation of hCx26-Hemichannels
Carsten Zeilinger and Melanie Steffens
Leibniz University Hanover, Institute of Biophysics
The human connexin 26 mediates ion transport from small to large molecules up to one kilodalton, which are essential for cell-cell communication and signal transduction. Under experimental conditions the voltage dependent conductance of the human connexon hCx26 could be measured after injection of a mixture of hCx26 mRNA and antisense of endogenous Cx38 (anti-Cx38) into Xenopus oocytes by voltage-clamp measurements. At 24–25°C voltage-clamp of oocytes at potentials above −40 mV evoked significant outward currents which were not observed in control oocytes. These currents were reversible amplified by increasing temperature of the bath solution and gradually reduced by decreasing bath temperatures below 20°C. In comparison the connexin rCx46 exhibited significant outward currents at all applied temperatures. To study this behaviour on purified protein, hCx26 was expressed in E. coli cells and folded into hexameric channel protein in presence of lipid. In lipid vesicles reconstituted hexameric purified hCx26was used to analyze channel function by flux measurements and lipid bilayer experiments. Since the Ca2+ sensitivity of hCx26 sustained during the temperature dependent activation, Lucifer Yellow (LY) was locked in the lipid vesicles in presence of micromolar Ca2+ concentrations. When a calcium chelator (EDTA) was added and temperature elevated above 23°C, LY was released from vesicles with reconstituted purified and hexameric hCx26 hemichannels. This approach allows the analysis of the channel activity by measuring transport rates, substrates and inihibitors using microtiter arrays and enables the estimation of the half maximal inhibition concentration of specific inhibitors like carbenoxolone or divalent cations. The inhibitory effects comparable to concentration as found by other methods. It is suggested, that this application can be used in general for analysis for agent screenings directed against to the channel function.
Poster Session 3.3: Connexins and Pannexins in Skin and Sensory Organs
3.35 A Novel Murine Living Skin Equivalent Amenable to Live Cell Imaging: Analysis of the Roles of Connexins in the Epidermis
Eve Kandyba, Dr Patricia Martin, and Dr Malcolm Hodgins
Glasgow Caledonian University
3D organotypic models are increasingly used to study aspects of epidermal organisation and cutaneous wound healing events. However, these are largely dependent on laborious histological analysis and immunohistochemical approaches. Despite the large resource of transgenic and knockout mice harbouring mutations relevant to skin disorders, few organotypic mouse skin models are available. We have developed a robust in vitro 3D organotypic mouse skin equivalent that reflects epidermal organisation in vivo. The system is optically transparent and ideally suited to real time analysis using a variety of integrated in situ imaging techniques. As a paradigm for co-ordination of cellular events, the epidermal gap junction network was investigated and the model displayed predominant Cx43 expression in basal proliferating cells and Cx26 and Cx30 expression in differentiated keratinocytes. We show that attenuation of Cx43-mediated communication by a connexin mimetic peptide enhanced wound closure rates in keratinocyte monocultures, emphasising the utility of the model to systematically unravel the molecular mechanisms underlying epidermal morphogenesis, assess novel therapeutic strategies and reduce any requirement for animal experimentation. Furthermore, we visualise epidermal regeneration following injury in real time, thereby facilitating avenues to explore distinctive modes of wound re-epithelialisation in a non-invasive manner. We now aim to use this model to begin to dissect the role of connexins in epidermal organisation and wound healing events by using keratinocytes isolated from Cx transgenic (Cx26D66H and Cx26G12R) and knockout (Cx43, 30 and 31) mice resources. Data on our wild type and connexin modified models will be presented.
3.36 Connexin 43 Acts As a Pro-inflammatory Mediator in Lungs
Maya Richani Sarieddine1, Bernard Foglia2, Irène Garcia-Gabay3, Tecla Dudez2, Isabelle Scerri2, and Marc Chanson2
1Geneva University Hospitals; 2Department of Pediatrics, HUG, Geneva, Switzerland; and 3Department of Pathology and Immunology, Faculty of Medicine, Geneva, Switzerland
Recurrent infection with P. aeruginosa and lung inflammation is a hallmark of cystic fibrosis. Cx43 is involved in the inflammatory response of various tissues, including lungs. Here, we monitored Cx43 expression and lung inflammation evoked by repetitive intratracheal instillation (IT) of P. aeruginosa endotoxin (LPS) in mice homozygous (Cx43+/+) and heterozygous (Cx43+/−) for Cx43. LPS was instilled once (IT1) to induce a first inflammatory response followed by two additional treatments (IT2 and IT3) each at the end of the inflammatory period. Maximal neutrophil count was detected in bronchoalveolar lavages (BALs) 24 hours after IT (+24 h) while inflammation resolved within 120 hours (+120 h). The repeated LPS treatment progressively increased the number of neutrophils recruited in airways 24 hours after each IT (IT1+24 h = 535'350 cells, IT2+24 h = 783′000 cells and IT3+24 h = 1′520′000 cells, medians from 6-8 experiments). Interestingly, LPS treatments induced a low molecular weight (41 kDa) isoform of Cx43 at +24 h, which disappeared with resolution of the inflammatory response. The appearance of the 41 kDa isoform during inflammatory periods coincided with detection of Cx43 in alveolar septa. In contrast, Cx43 in alveolar septa was not induced by LPS treatments of mice protected from inflammation by invalidation of the TNF-alpha gene, indicating that expression of Cx43 in lungs is inflammation-dependent. To evaluate the involvement of Cx43 in lung inflammation, repetitive LPS instillation was performed on Cx43+/− mice. Cx43+/− lungs express half of the amount of Cx43 and exhibit modest induction of its 41 kDa isoform after IT. In these animals, the number of neutrophils recruited in airways remained constant upon repetitive LPS treatments, representing a protection to inflammation of 54% at IT3. These results indicate that Cx43 is not only augmented during lung inflammation but also acts as a pro-inflammatory mediator. Targeting Cx43 in alveolar cells may protect from progressive injury in chronic inflammatory diseases.
3.37 A Novel Missense Mutation in the GJB2 Gene, S183F, Causes a New Deafness-Keratoderma Phenotype
M.A.M. van Steensel1, E.A. de Zwart-Storm2, and M. van Geel2
1University Hospital Maastricht and 2Department of Dermatology, University Hospital Maastricht
Dominant heterozygous mutations in the GJB2 gene coding for connexin26 cause a multitude of syndromes characterized by deafness and skin symptoms. So far, most mutations have been found in the first intracellular and extracellular domains. They are generally associated with severe skin symptoms. One exception is F142L, located in the third transmembrane domain. This particular mutation causes a mucositis-deafness phenotype with dental cysts. We recently saw a mother and daughter with sensory deafness and subtle skin changes consisting of translucent polygonal papules on the palms and mild plantar hyperkeratosis. While the skin phenotype was strongly reminiscent of disorders such as focal acral hyperkeratosis and acrokeratoelastoidosis, the deafness prompted us to analyze GJB2. In both patients, we found a heterozygous mutation 548C > T leading to the subsitution of serine 183 with phenylalanine. The mutation was not present in 100 unrelated Dutch controls. Expression of a GFP fusion construct in HeLa Ohio cells showed a pronounced transport defect, with prominent perinuclear localization of the mutant protein suggestive of accumulation in the ER. To our knowledge, this is the first report of a mutation in GJB2 affecting the last transmembrane domain. The mild skin phenotype is suprising. The transport defect is not more pronounced than that found for mutations such as N14K that cause more prominent skin symptoms, suggesting that transport defects may be epiphenomena not related to disease severity. Our findings might indicate that the fourth transmembrane domain of connexin26 is not as important for its function in the skin as the first intracellular and extracellular domains.
Poster Session 3.4: Pharmacology of Gap Junctions
3.39 Synchronization of Glucose-Induced Ca2+ Oscillations Evaluates Coupling Between Pancreatic Beta Cells
Sabine Bavamian1, Eliana Scemes2, Anne Charollais3, Serguei Startchik4, and Paolo Meda3
1CMU; 2Department Neurosciences, Albert Einstein College of Medicine, New York, USA; 3Department of Cell Physiology and Metabolism, University of Geneva School of Medicine, Geneva, Switzerland; and 4Bioimaging Core Facility, University of Geneva, Geneva, Switzerland
Pancreatic beta cells, which secrete insulin and are central to the development of diabetes, express Cx36. We have demonstrated the contribution of this connexin to beta cell function, notably in the intercellular synchronization of the [Ca2+]i transients generated during glucose stimulation. As a result, cells lacking Cx36 do not release insulin in a normal pulsatile fashion. Since this alteration is a landmark of human prediabetes, alterations in Cx36 signalling may play a role in the disease. If so, drugs acting on Cx36 channels may be therapeutically useful. Screening for such drugs is hindered by the difficulty to rapidly and non invasively evaluate coupling in large cell numbers. To address this issue, a high throughput model is required. We show that 1) MIN6 cells express Cx36, and feature a stimulus-secretion coupling and a Cx36-dependent synchronization of glucose-induced Ca2+ transients like primary beta cells; 2) MIN6 cells are adaptable for the automatic and non invasive recording of surface binding of many molecules; 3) after Fluo-3 loading, the same cells can be also studied fluorometrically for the intercellular synchronization of Ca2+ oscillations; 4) in the presence of glucose, most wild type MIN6 cells (which expressed levels of Cx36 as seen in native pancreatic islets) featured synchronous Ca2+ oscillations, whereas most of the MIN6 cells with reduced levels of Cx36 (after transfection of a Cx36 antisense cDNA) featured either asynchronous or nil Ca2+ oscillations; 5) glibenclamide, a sulphonylurea which enlarges beta cell gap junctions, increases the proportion of cells featuring synchronized Ca2+ waves. The data show that the intercellular synchronization of Ca2+ oscillations is a solid method to evaluate the coupling of the small diameter and high input resistance MIN6 cells, which can be used to identify novel, Cx36-targeted drugs of potential value in the therapy of diabetic dysfunctions.
3.40 2,2′,4,4′,5,5′-Hexachlorobiphenyl Modulates Turnover of Connexin43 in Rat Liver ‘Stem-Like’ WB-F344 Cells
Miroslav Machala1, Pavlina Polaskova1, Katerina Pencikova1, Pavel Krcmar1, Zdenek Andrysik2, and Jan Vondracek2
1Veterinary Research Institute, Brno, Czech Republic and 2Institute of Biophysics and Veterinary Research Institute, Brno Czech Republic
2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153), a widespread environmental pollutant, has been previously found to induce both acute and sustained inhibition of GJIC in a model of rat liver progenitor cells, WB-F344 cell line [Toxicol. Sci., 76, 102–111 (2003)]. In the present study, we examined effects of exposure to PCB 153 on connexin43 (Cx43) mRNA levels, phosphorylation and degradation of Cx43 protein, presence of gap junction plaques, and on transport of Cx43 from the endoplasmatic reticulum (ER) the Golgi apparatus (GA) in WB-F344 cells. While levels of Cx43 mRNA were not significantly affected by PCB 153, this PCB congener decreased both size and number of gap junction plaques and it potentiated internalization and degradation of Cx43 protein. Although ERK1/2 MAP kinases were activated by PCB 153, we found no evidence of Cx43 hyperphosphorylation, which would be related to inhibition of GJIC. However, PCB 153 increased phosphorylation of Cx43 after pretreatment with MG132, inhibitor of proteasome. This seems to suggest that PCB 153-induced phosphorylation of Cx43, mediated by ERK1/2, might contribute to Cx43 internalization and proteasomal degradation. Using brefeldin A in order to inhibit Cx43 transport from the ER to the GA, we observed that PCB 153 significantly delayed restoration of gap junction plaques in brefeldin A-pretreated cells. This suggested that PCB 153 might also interfere with process of Cx43 transport and gap junction assembly. Taken together, these data suggest that PCB 153 might affect several steps in the life cycle of Cx43 leading to a sustained downregulation of GJIC. This mode of action might contribute to its known tumor-promoting effects in liver cells. [Supported by the Czech Ministry of Agriculture, grant No. MZE0002716201 and the Czech Science Foundation, grant No. 524/06/0517.]
3.41 Acute Versus Chronic Regulation of Cardiac Gap Junction Channels by Phenylephrine and Isoprenaline
Jan Sebastian Schulte1, Aida Salameh2, Diana Rojas-Gomez3, Anja Scheffler3, F.W. Mohr3, and Stefan Dhein3
1Institute of Pharmacology and Toxicology, University of Münster; 2Clinic for Pediatric Cardiology, University of Leipzig; and 3Clinic for Cardiac Surgery, University of Leipzig
In pathophysioloy of cardiovascular disease elevated catecholamine levels play an important role. Furthermore changes in intercellular coupling by gap junction channels are often found in heart disease potentially affecting the network heart. Therefore we wanted to investigate the chronic effect of Phenylephrine (α -stimulation) or Isoprenaline (β -stimulation) on cardiac gap junction conductance and compare it to the acute effect of these substances. Cultured neonatal rat cardiomyocytes were incubated for 24 hours with 100 nM Phenylephrine or 100 nM Isoprenaline. Using the double-cell switch-clamp technique initial gap junction conductance of cell pairs was measured in absence of pharmaca (Tyrode's solution superfusion 1 ml/min, 37°C). For investigating acute effects unincubated neonatal rat cardiomyocytes were superfused with standard Tyrode's solution for a control phase (20 minutes). Then Phenylephrine or Isoprenaline was washed in (100nM, reaction time 20 minutes) and washed out afterwards. Gap junction conductance was measured once per minute during the whole experiment. Initial macroscopic gap junction conductance was 29+/−4 nS in unincubated cell pairs and significantly increased to 43+/−4 nS in cell pairs incubated 24 h with 100 nM Phenylephrine and to 63+/− 9 nS in pairs incubated with 100 nM Isoprenaline. These findings were underlined by western blot analyses showing selective increase in Cx43 expression after 24h adrenergic stimulation. In acute experiments gap junction conductance was significantly reduced by acute treatment with 100 nM Phenylephrine but left unaffected by treatment of 100 nM Isoprenaline. In conclusion acute and sub chronic effects of Phenylephrine and Isoprenaline on gap junctions in neonatal rat cardiomyocytes are different. Isoprenaline showing no significant acute effect on gap junction conductance leads chronically to an elevation of conductance (via Cx43 up regulation). Phenylephrine which acutely even reduces gap junction conductance also leads to conductance increase after long-term stimulation (also Cx43 up regulation). Last could even refer to a counter regulatory principle.
3.42 Agonist-Induced Inhibition of Gap Junctional Communication and Cx43 Dephosphorylation in Astrocytes
Martine Tence1, Edwige Amigou1, Pascal Ezan1, Eric Etienne2, and Christian Giaume1
1INSERM U840 and 2INSERM U833
Sphingosine-1-phosphate (S1P) and endothelin-1 (ET-1) are two mediators that increase in brain at sites of injury and inflammation. We have previously reported that they induce a rapid and strong inhibition of gap junctional communication (GJC) and increase the nonphosphorylated (NP) form of connexin43 (Cx43) in cultured mouse astrocytes. Here, we studied whether similar mechanisms underlie these effects and investigated whether Cx43 is associated with scaffolding proteins proposed to be involved in the regulation of GJ channels. For this purpose, we used the scrape-loading dye-transfer technique, Western blots and immunocytochemistry. Experiments with pertussis toxin, Clostridium difficile toxin B and Y27632 indicated that S1P- or ET-1-induced inhibition of GJC resulted from the activation of Gi/o- and Rho GTPase/ROCK-mediated intracellular pathways, both acting in a cooperative manner, the relative importance of each cascade being dependent on the mediator. The increase of Cx43 NP reflected a dephosphorylation of Cx43 engaged at junctional plaques and was impaired by cyclosporine or FK-506, suggesting a key role for the phosphatase PP2B. This process required Gi/o activation but neither ERK nor p38 MAPK. Both agonist-induced GJC inhibition and Cx43 dephosphorylation were independent on the cytoskeleton architecture, as indicated by the lack of effect of drugs that disrupt actin or microtubules. Confocal microscopy showed that astrocytes expressed ZO-1 and occludin, both displaying rings at cell-cell contacts which were colocalized with Cx43. Agonist-induced Cx43 dephosphorylation did not change this cellular distribution. Such associations were also observed in astrocytes submitted to metabolic inhibition, a treatment that totally disrupted GJC and massively increased Cx43 NP. This latter process involved a mechanism independent on Gi/o and required the combined actions of PP2B and PP1/PP2A. Altogether, these results indicate that different mechanisms of GJC inhibition and Cx43 dephosphorylation co-exist, the ET-1- and S1P-mediated mechanisms being dependent on the activation of several G proteins.
3.43 The Relationship Between TCDD-Induced Disruption of Contact Inhibition and Gap Junctional Intercellular Communication in Rat Liver ‘Stemlike’ Cells
Jan Vondracek1, Zdenek Andrysik1, Pavlina Polaskova2, Alois Kozubik1, and Miroslav Machala2
1Institute of Biophysics, Brno; and 2Veterinary Research Institute, Brno
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental toxicant and carcinogen, which is known to act primarily through activation of the aryl hydrocarbon receptor (AhR). Activation of AhR has been shown to be associated both with regulation of expression of xenobiotic-metabolizing enzymes, and with deregulation of apoptosis and cell proliferation in a cell-specific manner. TCDD is known to disrupt contact inhibition in rat liver WB-F344 cells, an in vitro model of bipotent liver progenitor cells, which consequently leads to enhanced cell proliferation. Such mechanisms might participate in tumor promoting effects of TCDD in liver, similar to defective connexin expression or connexin-mediated communication. Because cx43 has been shown to participate in regulation of cell proliferation, we investigated in the present study the effects of TCDD on GJIC in contact-inhibited cells. We found that TCDD induced AhR-dependent degradation of connexin 43 (cx43) protein and reduction of number of gap junction plaques in WB-F344 cells, which was accompanied with partial inhibition of GJIC, as determined by scrape loading/dye transfer assay. In contrast, TCDD had no effect on cx43 mRNA levels, and it did not affect phosphorylation of sites targeted by known kinases in C-terminal part of cx43 protein. Using cells stably transfected with dominant negative mutant of AhR, it was confirmed that effects of TCDD on cx43 are AhR-dependent. Therefore, these results suggest that AhR ligands might disrupt both GJIC and cell proliferation in rat liver ‘stem-like’ cells. Such effects might contribute to their known hepatocarcinogenicity. [This study was supported by the Czech Science Foundation (grant No. 524/06/0517) and the Czech Ministry of Agriculture (No. MZE0002716201).]
Index of Participating Authors
Please note that only authors/co-authors of oral abstracts and conference participants are listed. Coauthors on poster abstracts are not listed.
A
Alcolea, Sebastien, 282
Alessandro, Capponi, 282
Alonso, Florian, 283
Ambrosi, Cinzia, 279, 290
Angelillo-Scherrer, Anne, 284
Auerbach, David, 298
B
Bai, Donglin, 277, 279
Bargiello, Thaddeus A., 291
Bechberger, G. R., 288
Bechberger, John F., 289
Bechberger, John, 288
Bennett, Michael, 275
Bhalla, Ruchi, 279
Bassa, Daniela, 279
Brink, P. R., 284
Brown, Joshua, 290
Bruce, Alexandra, 286
Bunse, Stefanie, 295
Burra, Sirisha, 278
Burt, Janis, 292
Butera, John, 298, 299
C
Capponi, A., 283
Chandrasekhar, Anjana, 286
Chanson, Marc, 284
Ciaccio, Edward J., 285
Cina, Cima, 289
Civitelli, Roberto, 287
Claassen, Yvonne, 290
Cohen, I. S., 284
Coombs, Wanda, 298
Coromilas, James, 285
Cowan, Bryce J., 293
Cowan, Kyle N., 293
D
Dahl, Gerhard, 277, 279, 279
Dai, Ping, 295
de Diego, Alberto Cabestrero, 294
de Wit, Cor, 282
Delmar, Mario, 298
Dermietzel, Rolf, 295
Derouette, Jean-Paul, 276
Desplantez, Thomas, 276
Dhein, Stefan, 296
Dias, Priyanthi, 294
Dobrowolski, Radoslaw, 287
Duffy, Heather S., 285
Dunn, Clarence A., 280
Dupont, Emmanuel, 286, 294
F
Fontana, Pierre, 284
Fujiyoshi, Yoshinori, 291
G
Gaietta, Guido, 279
Gong, Xiang-Qun, 279
Gordon, C., 284
Gossman, David G., 278
Gourdie, Robert, 281
Green, Colin R., 289
Gros, Daniel, 282
Gu, Sumin, 278
Guillotin, Bertrand, 287
H
Haefliger, J.-A., 283
Haefliger, Jacques-Antoine, 282
Harris, Andrew L., 297
Harris, Stephen E., 278
Haugan, Ketil, 299
Hennan, James K., 299
Heyman, Nathanael, 292
Hiroaki, Yoko, 291
Hirst-Jensen, Bethany J., 293
Hunter, Andrew, 281
J
Jalife, José, 298
Jiang, Jean, 278, 296
Jin, Chengshi, 281
K
Kamermans, Maarten, 290, 295
Kanaporis, G., 284
Kantrowitz, Joel, 299
Keane, Robert W., 279
Kellezi, Admir, 293
Kidder, Gerald M., 277
Kieken, Fabien, 293
Kinoshita, Shigeru, 295
Klooster, Jan, 290, 295
Knudsen, Carsten Boye, 298
Kozoriz, M., 288
Krattinger, N., 283
Kurata, Wendy, 281
Kwak, Brenda R., 276
Kwak, Brenda, 284
L
Laird, Dale W., 279, 287, 293
Lampe, Paul, 280
Langlois, St'ephanie, 293
Larsen, Bjarne D., 298
Larsen, Bjarne Due, 299
Lau, Alan, 281
Lewandowski, Rebecca, 298
Li, Tony Y., 277
Li, Xinli, 281
Lin, Xianming, 300
Liu, Jialu, 296
Liu, Kun, 299
Locke, Darren, 297
Locovei, Silviu, 277, 279
Lounsbury, Crystal S., 287
M
Ma, Meiyun, 279
Maass, K., 288
Macleod, Kenneth T., 294
Mazzolai, L., 283
Mazzolai, Lucia, 282
McCulloch, Fiona, 275
Meda, P., 283
Meda, Paolo, 282
Mironov, Sergei, 298
Morgan, Gwen, 299
Muñoz, Viviana, 298
N
Nakano, Yukiko, 295
Nathalie, Krattinger, 282
Naus, C. C., 288
Naus, Christian C. G., 289
Naus, Kathryn E., 277
Nicholson, Bruce, 286, 292
Nicholson, Louise F. B., 289
Nicod, P., 283
Nicod, Pascal, 282
O
O'Carroll, Simon J., 289
Ornitz, David, 287
Oshima, Atsunori, 290, 291
Osorio, Juan Carlos, 285
Oyamada, Masahito, 295
P
Penuela, Silvia, 279
Petersen, Jorgen S., 299
Petersen, Jørgen Søberg, 298
Peti-Peterdi, Janos, 275
Plante, Isabelle, 287
Prochnow, Nora, 295
Q
Qiu, Feng, 277
R
Robinson, R. B., 284
Rosen, M. R., 284
Roth, Isabelle, 276, 284
Rothery, Stephen, 286
Rowlinson, Giselle, 294
S
Saez, Juan Carlos, 275
Scemes, Eliana, 277
Schmid, Michael, 290
Schmidt, Volker, 282
Schweda, Frank, 282
Severs, Nicholas J., 286, 294
Shao, Qing, 279, 293
Shibayama, Junko, 298
Shields, Colleen, 290
Siller-Jackson, Arlene J., 278
Simon, A., 283
Sipos, Arnold, 275
Sjoerdsma, Trijntje, 290
Smock, Amy, 290
Solan, Joell L., 280
Sorgen, Paul, 293
Sosinsky, Gina E., 291
Sosinsky, Gina, 279, 290
Sprague, Eugene, 278
Spray, David, 277
Srinivas, Miduturu, 291
Su, Vivian, 281
Suen, M., 288
Sugamele, Rocco, 284
Swillo, Robert, 299
T
Taffet, Steven, 298
Takamatsu, Tetsuro, 295
Tang, Qingxiu, 291
Tani, Kazutoshi, 291
Tao, Liang, 297
Thomas, Neil, 294
Toloue, Masoud, 292
Tong, Dan, 277
Trelles, M. Pilar, 291
V
Valiunas, Virgis, 284
Valiuniene, L., 284
Veenstra, Richard, 300
Verselis, Vytas, 291
W
Wölfle, Stephanie, 282
Wagner, Charlotte, 282
Wang, Junjie, 279
Watkins, Marcus, 287
Weingart, Robert, 276
Willecke, K., 288
Willecke, Klaus, 287
Wong, Cindy, 276
Z
Zhao, Hong-Bo, 278
Zoidl, Georg, 295
Zucker, Shoshanna N., 286
Zuckerman, J., 284