Abstract
This synopsis covers the main results and conclusions from the platform presentations during the International Gap Junction Conference. More detailed information is provided in the mini reviews on controversial scientific issues, short reports of research results and conference abstracts published in this issue of Cell Communication and Adhesion.
The conference took place in the comfortable hotel Marienlyst located north of Copenhagen in the small town of Elsinore, close to the waterfront of the Kattegat sea. As planned, this setting facilitated intensive scientific discussion during the oral and poster sessions, that were separated by enjoyment of the beautiful scenery and the sunny weather outside during the breaks.
Instead of having one keynote speaker on the opening night as in previous years, Michael Bennett (hemichannels), Brian Duling (connexins in vascular physiology), Maiken Nedergaard (connexins in astrocytes) and Stefan Dhein (pharmacology) were asked to give longer overview talks to open discussion of selected fields. We would like to take this opportunity to thank the speakers for presenting excellent perspectives on the status within their research fields.
During the conference differences in perspectives and interpretation of data became evident. This included discussion controversies in subjects such as the physiological role of hemichannels, the utility of connexin-mimetic peptides and the role of connexins in vascular function. In this issue, we refer readers to two discussion papers that consider the mechanism of action and possible utility of mimetic peptides.
The scientific program started with a lecture on connexin hemichannels by Mike Bennett. In collaboration with the group of Juan Saez, he reported that lowering the intracellular redox potential with dithiothreitol increased Cx43 hemichannel opening and altered paracrine signalling as well as intercellular interactions. Thus, Mike concluded, hemichannels played an important physiological role; he ended with the statement “the sun is rising on hemichannels”. Not all the speakers of the following two sessions on hemichannels appeared to agree with this general statement but the majority of their results were interpreted to be due to hemichannel activity. Hemichannels were suggested to be involved in several physiological processes: for example, ATP from renal epithelial cells might be released via Cx30 hemichannels (Janos Peti-Peterdi et al.) and via Cx43 hemichannels from ovarian granulosa cells (Dan Tong et al.). Mutated Cx37 hemichannels appear to be involved in the initiation of artherosclerotic plaque formation (Jean-Paul Derouette et al.), and Hong-Bo Zhao and David Gossmann reported that hemichannels mediate release of inositol-1,4,5 trisphosphate in the cochlea.
The discussion of pannexin function was initiated by Gerhard Dahl et al. who concluded that pannexin1 is part of the pore forming unit of the P2x7 receptor death complex. Two groups (Daniela Boassa et al. and Silvia Penuela et al.) presented evidence that pannexins 1 and 3 are glycosylated proteins, a finding that contrasts with connexin proteins which are apparently not glycosylated. These findings could possibly explain the published results that pannexins can form gap junction channels in Xenopus oocytes, where they are presumably not glycosylated, but do not do so in cultured mammalian cells as tested by several investigators. Furthermore, Dahl et al. found that connexin-mimetic peptides used at low concentrations inhibited pannexin channel currents but not connexin channel currents. Previous studies from other laboratories had indicated at least some specificity of the connexin-mimetic peptides towards their connexin protein targets. In general, the discussion on pannexin hemichannels further clouded the discussion of the physiological role of connexin hemichannels and made it clear that investigators need to utilize careful experimentation to distinguish the source of hemichannel activity.
Ross Johnson et al. studied the uptake of fluorescent dyes into cultured cells and found two distinct classes of dye uptake; the first class involved pannexin1 protein and the second class involved Cx43. Elena Scemes et al. showed that several pharmacological compounds originally described as blocking connexin channels also inhibit pannexin hemichannels and thus cannot be used to distinguish between these different kinds of hemichannels.
In the session on the life cycle of connexins, Paul Lampe et al. introduced new antibodies which specifically recognize phosphorylation sites at positions S365, S368 and S325/328/330 in the Cx43 C-terminal region. These appear to be very useful tools to unravel the contribution of these phosphorylation sites to wound healing, myocyte hypoxia and development. First results suggest that different kinases phosphorylate several amino acid residues thereby altering the conformation of the Cx43 C-terminal region.
Several groups investigated binding proteins to Cx43. Michael Koval et al. identified ERp29, a thioredoxin-fold protein in the endoplasmic reticulum (ER) that appears to restrict disulfide bond formation between the two extracellular loop domains of Cx43. Vivian Su et al. reported on another Cx43-binding protein, CIP75, which contains a ubiquitin-like domain and a ubiquitin-associated domain. The authors showed that CIP75 interacts with components of the proteasome and thus is likely to have a role in the ER-associated degradation of Cx43.
The association of Cx43 with ZO1 was further explored by Andrew Hunter and Robert Gourdie. Using imaging techniques, they found that (tagged) N-cadherin is also located in this complex that appears to affect turnover of Cx43-containing gap junctions by modulating linkages to the actin cytoskeleton. Furthermore, Spray et al. presented evidence that the Cx32 protein needs microtubule-dependent motor proteins (e.g., kinesin), in order to get to its location inside the cell.
Surprisingly, few contributions were devoted to innexins, i.e., gap junction proteins in invertebrates which are analogous but do not show any sequence identity to the connexins in vertebrates. Using Drosophila embryos, Hildegard Lechner and Michael Hoch found that innexin2 is regulated by the paracrine protein factors hedgehog and wingless. In turn, innexin2 affects the transcription of the corresponding genes. The molecular mechanism of this intricate regulatory network remains to be worked out. Michael Hoch et al. presented further evidence for the involvement of innexins in the polarity of Drosophila larval proventricular epithelium. In innexin2 mutants, innexin1 and innexin3 are also transcriptionally downregulated. This affected the multilayering of the proventricular epithelium. How gap junction channels built of innexins or connexins affect transcription of other genes is neither understood in invertebrates nor in vertebrates and appears to be a formidable task for future efforts in this research field.
Three sessions of the conference were devoted to connexins in the cardiovascular system, reflecting the large impact of this area on gap junction research. In his opening lecture, Brian Duling presented an excellent overview on gap junctions in the vascular wall. His group used Cx40- and Cx43-deficient mice and found that both connexins contribute to the propagation of action potentials along endothelial cells in arterioles. How ion channels–in analogy to nerve conduction–affect the propagation of action potentials along the vascular wall is still a matter of debate and an important research area for future investigations. In addition, the role of gap junctions in endothelium and smooth muscle cells is still not clear. For example, the functional contribution of Cx45 in vascular smooth muscle cells is not yet known, but Cor de Wit et al. showed that replacing Cx40 with Cx45 partially inhibits the blood pressure increase seen in Cx40KO mice. In contrast it does not restore the ability to conduct vasodilations. Furthermore, both de Wit et al. and Jacques-Antoine Haefliger et al. found that Cx40KO mice had increased renin levels due to abnormal regulation of renin release, and that inhibition of the renin–angiotensin system could either partly or fully prevent hypertension in Cx40KO mice. Hypertension was also shown to induce remodeling of the aorta, and Florian Alonso et al. demonstrated that hypertension profoundly affected connexin expression. Alexander Simon et al. analyzed lymphatic defects in mice deficient for Cx37 and Cx40. In addition to their role in stabilizing blood vessels, these two connexins are also required for the development and function of central lymphatic trunks, such as the thoracic duct. Cx37–/–, Cx43+/– mice develop a milky effusion in the pleural cavity and edemas.
Brenda Kwak et al. presented data showing that Cx37 plays a role in hemostasis. Cx37KO mice showed significantly accelerated platelet plug formation, and several lines of evidence supported a role for Cx37 in inhibition of platelet aggregation. Using high-resolution optical mapping, Gregory Morley and Pamela Riva demonstrated that Cx40 plays a key role in stabilizing the SA node as the primary pacemaker. Their data indicated that the absence of Cx40 is associated with multiple ectopic pacemaker sites, a condition that develops between murine embryonic day E13.5 and E15.5. In a model system for pacemaker function, Virgis Valiunas et al. quantitated the coupling needed for functional interaction between cardiomyocytes and artificial pacemaker cells. These interactions were shown to be necessary for both hyperpolarization of the pacemaker cells (to activate pacemaker currents) and subsequent depolarization of the cardiomyocyte.
Mutations in Cx43 causing oculodentodigital dysplasia (ODDD) are only rarely associated with a cardiac phenotype in humans. However, Radoslaw Dobrowolski et al. showed that in transgenic mice carrying the Cx43G138R (ODDD) mutation in vitro and in vivo, arrhythmias were observed which appeared to be aggravated by hemichannel activity. The role of hypoxia in regulation of gap junctions was the subject of the subsequent talk by Heather Duffy et al., showing that hypoxia regulates Cx43 expression and localization via HIF-1α. The associated changes in Cx43 were shown to induce conduction slowing in the infarct border zone and are thus likely to increase the risk of developing arrhythmia. Remodeling in human heart failure was shown to be associated with increased interaction between Cx43 and ZO-1, as shown by Alexandra Bruce and colleagues. ZO-1 has been associated with regulation of plaque size and may explain the reduced size observed in heart failure. Glenn Radice et al. reported that a reduction in plaque size and Cx43-phosphorylation occurred in mice haploinsufficient in N-cadherin. These changes were associated with increased susceptibility to arrhythmia, indicating that the mechanical stability of the intercalated disc is essential for coupling efficacy. This was further stressed in the two following talks investigating the role of plakophilin in regulating Cx43. Rosy Joshi-Mukherjee et al. showed that Cx43 can be co-precipitated with plakophilin and vice-versa. Using surface plasmon resonance it was demonstrated that plakophilin is able to bind to the C-terminus, indicating direct interaction. Mutations in plakophilin are associated with arrhythmogenic right ventricular dysplasia (ARVD), and Eva Oxford et al. showed that silencing of plakophilin is a sufficient signal to reduce and redistribute Cx43. This could represent an important mechanism in creating arrhythmogenic substrates in ARVD.
Two talks highlighted the role of Cx43 in bone formation in mice and zebrafish. Roberto Civitelli et al. used transgenic mice with specific deletion of Cx43 in chondro-osteoprogenitor cells and found that these mice were smaller and had thinner bones. The size of the growth plates was decreased by one-third. Again, the detailed molecular mechanism is not clear, although a model was suggested. Kathy Iovine et al. studied skeletal malformations in the fins of zebrafish due to Cx43 mutations. They noticed defects in cell proliferation and segment length. Since Cx43 (at least in mammals) has several essential functions in different organs, it may be necessary in the future to restrict the range of Cx43 mutations also in zebrafish to certain cell types. Jean Jiang et al. concluded from their results that the release of prostaglandin E2 from established oocytes due to mechanical stress occurs via Cx43 hemichannels which appear to be associated with α 5 integrin.
Isabelle Plante et al. reported that the Cx43G60S point mutation, as a mouse model for oculodentodigital dysplasia, caused delayed development of the mammary gland and defects in the expulsion of milk. Julia Hatler et al. have found that morpholino-mediated knock-down of Cx43.4 in zebrafish altered in part the left–right asymmetry of the developing heart. The authors hypothesized that Cx43 is required to maintain epithelial cell integrity. Bruce Nicholson et al. studied the effect of different Cx26 mutations on several of the transformed phenotypes in transfected HeLa cells. cAMP appears to pass through Cx26 gap junction channels more easily than through Cx43 or Cx32 channels. The authors concluded that the ability of Cx26 channels to transfer cAMP is responsible for its growth-suppressing effect.
Two sessions of the conference were devoted to connexin expression and function in nervous tissue. Maiken Nedergaard presented a very impressive overview lecture on multiple roles of connexins in astrocytes. Using two-photon laser scanning microscopy she has described an astrocytic Ca2 + increase after whisker stimulation in wild-type and Cx43-deficient mice. Based on electrophysiological evidence, she concluded that Cx43 hemichannels in astrocytes are permeable to ATP. Furthermore, her group studied the fate of fetal human astrocytes in the mouse brain nine months after injection. Apparently, Ca2 + waves can be propagated three-fold faster by human astrocytes than by mouse astrocytes. This was most likely due to the larger size of the human astrocytes. John Bechberger et al. have investigated stroke induced by occlusion of the middle cerebral artery in mice. They found that the damaged brain area is increased in mice lacking astrocytic Cx43 or the C-terminus of Cx43. Simon O'Carroll et al. have studied the spread of damage after spinal cord injury in rats and concluded that a mimetic peptide to Cx43 can prevent swelling, neuronal cell death and astrogliosis by inhibiting the opening of Cx43 hemichannels.
A number of presentations were focused on the ongoing quest to determine the role of connexins in brain development. Deletion of Cx26- and Cx43-coding DNAs from radial glia cell precursors, using appropriate cell type–specific transgenic mice, altered normal lamination of the intermediate zone and the cortical plate, as shown by Cina et al. It should be interesting to extend these studies to other connexins and investigate their role, if any, on neural migration in the brain. Catherine Sorbara et al. described that Cx36 deletion in neural progenitor cells increased cell proliferation in the subventricular zone of the dentate gyrus and concluded that Cx36 controls proliferation of doublecortin-expressing neuroblasts. Julia von Maltzahn et al. have generated a new transgenic mouse in which the Cx45 protein is tagged by EGFP. These mice should be useful for investigating the location of Cx45 in certain neurons and identification of these cells in acute brain slices. The physiological role of neuronal connexins, other than Cx36 (e.g., Cx45 and Cx30.2) remains elusive so far.
The role of connexins in vision was represented in several talks during the meeting and exciting new results were presented. David Paul et al. have generated a new transgenic mouse which specifically lacks Cx36 in cone photoreceptor cells of the retina. Their results suggest that Cx36 mediates rod–cone coupling, which appears to be required for vision under mesopic light conditions. Stephan Sonntag et al. have characterized α CaMKII binding and phosphorylation sites on the Cx36 protein which are very similar to the sites on the N-methyl-D-aspartate receptor subunit NR2B. They conclude that Cx36 could activate autonomous phosphorylation of α CaMKII, similar to the binding of NR2B to CaMKII. These interactions could modulate synaptic transmission and might be related to the consolidation of memory.
Maarten Kamermans and his associates have generated zebrafish with point mutations in Cx55.5, which is specifically expressed in horizontal cells. The homozygous mutants exhibit reduced sensitivity towards the optokinetic response. The authors interpret their results to indicate that Cx55.5 hemichannels provide ephaptic feedback of horizontal cells to cone photoreceptor cells within the restricted area of cone pedicles. Georg Zoidl et al. presented evidence that pannexin1 is expressed in zebrafish horizontal cells. Using specific antibodies they found Cx55.5 and pannexin1 at different locations in horizontal cells of cone pedicles.
This year's conference offered a number of excellent presentations in the sessions on channel structure and function. Work from Gina Sosinsky and coworkers involving four different Cx26 mutants, shed new light on the role of amino acids involved in interactions between protein subunits. One of the Cx26 mutants (hCx26M34A) that showed decreased activity, but not unstable interactions, was suggested by Atsunori Oshima et al. to have a plug in the pore, which could possibly account for the decreased activity of this mutant.
Vytas Verselis and coworkers presented data that implicated specific amino acid residues of Cx50 present at the border between the first transmembrane and extracellular loop to be involved in voltage-dependent gating of Cx50 hemichannels. The importance of the N-terminus of Cx37 was pointed out by the presentation of Eric Beyer and coworkers. Using a number of overlapping deletions in the N-terminal domain, it was shown that none of the mutants formed functional hemichannels, although most deletions were tolerated in terms of trafficking and plaque formation. This effect was dominant in cotransfections with WT-Cx37.
Two talks addressed the issue of permeability of gap junction channels to different metabolites and dyes. Massoud Toloue and Bruce Nicholson presented a method of measuring permeability of ATP, AMP and cAMP in Cx26, Cx32 and Cx43 channels. The results demonstrated large differences in permeability that could affect the degree of metabolite sharing in tissues with different connexin composition. The complexity is further increased by the fact that permeability to small ions (current) and metabolites (and dyes) can be regulated individually, as reported by Nathanel Heyman and Janis Burt. They showed that most likely dye-permeable and dye-impermeable states exist, and that the Cx43 C-terminus and serine 368 are involved in the transition between states.
Several talks focused on the important direct interactions between connexins and other proteins. Elke Winterhager et al. showed that expression of Cx43 in carcinoma cells reduced their proliferation and upregulated the growth regulatory protein NOV. Cx43 colocalized with NOV via its C-terminus, which when expressed alone also reduced proliferation. Another interaction was presented by Stephanie Langlois et al., who showed that Cx43 interacts with caveolin via its C-terminus, both in the Golgi complex and at the plasma membrane. In addition they reported that intercellular coupling is dependent on the presence of caveolin, and that a reduction of caveolin reduces coupling. Furthermore, overexpression of caveolin in a caveolin-deficient cell line markedly increased coupling. Paul Sorgen et al. found that binding of c-Src to Cx43 does not itself disrupt the interaction between Cx43 and ZO-1. Rather, the SH3 domain of c-Src binds to the PDZ-2 domain of ZO-1 in a manner facilitated by the C-terminus of Cx43. Jiang et al. found a role for interacting proteins in the correct localization of gap junctions. They investigated the physiological role of interaction between aquaporin-0 and chicken Cx50. Exchange of the Cx50 intracellular loop (which is responsible for the interaction with aquaporin-0) with the loop of Cx46 led to prevention of proper trafficking and retention of the chimeric protein in the cytoplasm.
In the mammalian heart several connexins are coexpressed in varying amounts in different regions. In order to mimic this situation Priyanthi Dias et al. have coexpressed controlled amounts of different connexins in a rat liver epithelial cell line. Using this system they analyzed how different expression levels of transfected Cx40 or Cx45 affected intercellular coupling relative to endogenous Cx43. The predictions from these experiments fit well with data obtained in clones of the atrial cell line HL-1, which were selected for different conduction velocities.
Oyamada et al. have observed, using the rat scrape injury model of corneal endothelial injury, that knock-down of Cx43 by antisense or interference RNA accelerates wound healing, similarly to previously published reports for epidermal skin wounds. Thus, pharmaceutical compounds which inhibit Cx43 might be useful for quicker healing of wounds in the skin and in the cornea.
Paolo Meda et al. have studied Cx30-deficient mice which are deaf, similar to Cx30-deficient humans. Formation of the capillary network of the stria vascularis precedes the development of the endocochlear potential. The authors found a disrupted endothelial barrier in the capillaries, which could explain the loss of the endocochlear potential. Possibly, cochlear epithelial cells require Cx30 to produce a paracrine factor that controls the integrity of the capillary network in the stria vascularis.
The sessions on the pharmacology of gap junctions were introduced by Stefan Dhein, who gave an extensive overview of the current knowledge on drugs and other compounds that affect gap junctions. Many of the “classic” drugs acutely uncouple gap junctions, but some compounds that acutely increase coupling, among these the anti-arrhythmic peptide and its analogues, have been discovered. Apart from these acute regulators, many compounds and hormones regulate the expression of connexins and thereby affect coupling. Given the number of diseases where connexins have been implicated, there is great potential for developing new drugs that specifically target connexins.
Darren Locke et al. presented data that shed light on the mechanism underlying the sensitivity of Cx26 homomeric and Cx26/32 heteromeric channels to aminosulfonates. Tagging of the Cx26 C-terminus rendered the resulting hemichannels insensitive to aminosulfonates, implying the involvement of the C-terminus. These effects were not restricted to hemichannels, but were also observed with cell-to-cell channels.
One of the new pharmacological tools, the peptide “RXP-E”, was developed to specifically bind to the C-terminus of Cx43 and antagonize uncoupling of cells by heptanol and acidification. Rebecca Lewandowski et al. presented data that connecting RXP-E (which cannot permeate the plasma membrane) to specific amino acid sequences promotes the cellular uptake of the peptide in multicellular preparations. Using this approach, it was shown that RXP-E can interact with native Cx43 and thereby prevent conduction block in cardiac myocytes treated with heptanol.
The remaining presentations addressed derivatives of the anti-arrhythmic peptide (AAP). Patricia Martin and coworkers showed that AAP10 was able to upregulate expression and coupling via Cx40 and Cx43 in a PKC-dependent manner. In contrast, no effects were observed in Cx26-expressing cells. At the level of electrical coupling, Richard Veenstra et al. reported that rotigaptide significantly affected inactivation, by both slowing the rate of inactivation and reducing the level of inactivation. This could explain the effects of rotigaptide on slowed conduction and conduction block. Bjarne Due Larsen et al. presented the journey from AAP and its early derivatives to the more stable analogue, rotigaptide, which is currently used in clinical trials. Furthermore, using these drugs as templates, smaller compounds that can be taken up orally have now been developed; James Hennan et al. presented the first data on the anti-arrhythmic effects of these new compounds. The compounds have a profile similar to that of rotigaptide, prevented conduction slowing during metabolic stress and delayed conduction block after calcium infusion in mice. In dogs, reperfusion arrhythmias were significantly reduced as were the size of the experimental infarcts.
CONCLUDING REMARKS
After about 25 years of molecular research on gap junctions, Cx43 expression has finally been recognized as a potential target for therapeutic intervention via development of anti-arrhythmic drugs or pharmaceutical compounds to accelerate wound healing. Few connexin-altering compounds appear to be close to entering the pharmaceutical market. However, the development of useful drugs takes usually more than ten years after the target molecules have been identified.
Some areas of gap junction research were represented more or less for the first time in the history of the biannual gap junction conferences. For example, the involvement of connexins in the physiology of the kidney and in the function of the lymphatic system fall into this category. Whereas some progress has been achieved in understanding the functional interplay of cardiac connexin channels, many questions remain unanswered with regard to their functions in blood vessels. Furthermore, the contribution of connexins to the development and function of the brain is just beginning to be unraveled. Although the characterization of connexin-deficient mice had led us to assign functional categories to each member of the connexin gene family, one can expect further insights into the interaction of connexin isoforms and their binding proteins from the characterization of connexin point mutations in humans, mice and zebrafish. We still do not understand the molecular mechanisms underlying the phenotypic abnormalities observed in animals or humans with known disease-causing connexin mutations. Do pannexins form gap junction channels under physiological conditions, or do they only function as (hemi)channels connecting the cytoplasm of the cells with the surrounding medium? After this conference one gets the impression that the community of gap junction researchers is just beginning to unravel the delicate network of how connexins and/or pannexins are involved in intercellular signaling.
Due to space limitations for this synopsis we concentrated on the platform presentations, although many poster contributions were, in our opinion, of equal importance. We apologize to all colleagues whose work could not be covered in this review. We hope that their work will equally stimulate other researchers and that they have obtained useful feedback during this conference.