Regulation of Connexin43 Oligomerization is Saturable

Abstract

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.

Keywords:

• gap junction
• di-lysine motif
• assembly
• Golgi apparatus

INTRODUCTION

Gap junction proteins in the connexin family form channels that interconnect adjacent cells (1, 2, 3, 4, 5). A complete gap junction channel is formed when a connexin hexamer in the plasma membrane of one cell binds to a hexamer in an adjacent cell. Unlike most other multimeric transmembrane complexes, which typically oligomerize in the endoplasmic reticulum (ER) as a prerequisite to further transport along the secretory pathway (6), connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus (7, 8, 9, 10, 11). However, defining the mechanisms which regulate post-ER oligomerization of Cx43 has been a challenging problem in the field. In particular, mechanisms which prevent Cx43 oligomerization in the ER are not understood at a molecular level.

We have used connexins containing a di-lysine ER retention/retrieval motif (HKKSL) to study early events in connexin oligomerization (12, 13). HKKSL-tagged connexins are retained in the ER in the absence of pharmacologic agents that have the potential to alter ER composition and function (14, 15). Previously, we found that Cx43-HKKSL expressed by stably transfected HeLa cells is retained in the ER in an unoligomerized, monomeric state (12, 13). Here, we found that overexpressing Cx43-HKKSL caused it to escape from the ER oligomerize into hexamers, and form intracellular gap-junction-like structures. Thus, the capacity of HeLa cells to regulate Cx43 oligomerization was saturable.

MATERIALS AND METHODS

Antisera and Reagents

Rabbit anti-Cx43 was from Sigma (St. Louis, MO), mouse anti-Cx43 was from Chemicon (Temecula, CA). Mouse anti-calnexin, anti-vimentin, and rabbit anti-mannosidase II and anti-β -actin were from Affinity Bioreagents (Golden, CO). Fluorescent and horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Triton X-100 was from Roche Molecular Biochemicals (Indianapolis, IN). Tissue culture reagents were from Invitrogen (Carlsbad, CA). Unless otherwise specified, all other reagents were from Sigma.

Generation of Stable Transfectants

HKKSL-tagged Cx43 was prepared as previously described (12). DNA for transfection was purified from bacteria using the Qiagen Maxiprep kit according to the manufacturers instructions. Prior to transfection, cDNA was purified by EtOH precipitation. HeLa cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA), passed and stably transfected cells were selected using 2 mg/ml G-418 (Invitrogen). The cells were then plated into three pools using selective medium at low cell density and individual colonies were selected, expanded, and screened by immunofluorescence microscopy for Cx43-HKKSL expression. The clone numbering scheme denotes the pool number (1, 2 or 3) followed by the clone number. In this study, we analyzed clones 1(1), 1(6), 1(11) and 2(2) which were representative of stably transfected cells that had ER localized Cx43-HKKSL and clone 3(3) which was representative of stably transfected cells that had Cx43-HKKSL localized to a perinuclear intracellular compartment.

Immunohistochemistry

For immunofluorescence, cells plated on glass cover slips were fixed and permeabilized with MeOH/acetone (1:1), then washed 3x with PBS, followed by PBS + 0.5% Triton X-100 and PBS + 0.5% Triton X-100 + 2% goat serum (PBS/GS). The cells were incubated with primary antisera diluted into PBS/GS for 1 h, washed, and then labeled with secondary antisera diluted into PBS/GS. The cells were then washed with PBS, mounted into MOWIOL, visualized by fluorescence microscopy using an Olympus X-70 microscope system and imaged with a Hammatzu Orca-1 CCD camera and Image Pro image analysis software (Media Cybernetics, Silver Spring, MD).

Postembedding immunogold electron microscopy (EM) was done by the University of Pennsylvania Biomedical Imaging Core. Cells were fixed in 2.5% glutaraldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, and embedded and sectioned as previously described (7). Ultrathin sections were placed on nickel-coated grids and processed for immunostaining using a polyclonal antibody directed against Cx43 and secondary goat anti-rabbit IgG conjugated to 20 nm colloidal gold particles.

Protein Analysis

The techniques outlined below have also been described elsewhere (7, 10, 16). Cells were washed with PBS, harvested into PBS containing protease inhibitors (10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl chloride, 2 μ g/ml leupeptin and 1 μ g/ml pepstatin) and phosphatase inhibitors (1mM NaVO₄ and 10mM NaF) then passed through a ball bearing homogenizer 100 times (7, 10). The homogenate was centrifuged at 500 × g for 5 min using an IEC CL3R centrifuge and the resulting post nuclear supernatant was centrifuged at 100,000 × g for 30 min using a Sorvall Ultra Pro 80 ultracentrifuge to obtain a membrane enriched pellet. To analyze total cell connexin expression, this pellet was resuspended in SDS-PAGE sample buffer, resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and then blocked overnight with 40 mM TRIS, pH 7.5, 5% Carnation powdered milk, and 0.1% Tween 20 (Blotto). The blots were incubated with Blotto containing specific antisera, washed, and then further incubated with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson Immunoresearch, Malvern, PA). Specific signals corresponding to a given protein were detected with the enhanced chemiluminescence reagent (Amersham) and quantified with a Kodak EDAS system (Rochester, NY).

For detergent solubilization studies, the membrane enriched pellet was resuspended in PBS + inhibitors containing 1% Triton X-100 and then incubated for 30 min at 4°C. The sample was then centrifuged at 100,000 × g for 30 min and separated into Triton X-100 soluble supernatant and insoluble pellet fractions. The soluble and insoluble fractions were then resuspended into SDS-PAGE sample buffer, and assayed by immunoblot. For in situ Triton X-100 solubility, cells were incubated in PBS containing 0.675 mM CaCl₂, 0.2 mM MgCl₂ and 1% Triton X-100 for 30 minutes at 15°C, washed with PBS, fixed with methanol/acetone and analyzed by immunofluorescence microscopy as described above.

Sucrose gradient fractionation was done using post nuclear supernatants solubilized in 1% Triton X-100 for 30 min at 4°C. The samples were then centrifuged at 100,000 × g for 30 min and the resulting Triton X-100 soluble fraction was overlaid onto a 5–20% sucrose gradient. The gradient was centrifuged at 148,000 × g for 16 h at 4°C in a Sorvall Ultra Pro 80 centrifuge using an AH-650 swinging bucket rotor. Following centrifugation, 0.5 ml fractions were collected from the bottom of the centrifuge tube at 4°C, and then analyzed by immunoblot.

RESULTS

In contrast to wild type Cx43, which localizes predominantly to the plasma membrane, Cx43-HKKSL, which has an ER retention/retrieval signal, localizes to the ER (12, 13). In the course of using Cx43-HKKSL as a tool to study early events in connexin oligomerization, we generated a series of stably transfected HeLa cell clones. Most of these HeLa cell lines, including clones 1(1), 1(6), 1 (11), and 2(2), had Cx43-HKKSL localized to the ER (Figure 1). However, some stably transfected cell clones, such as the 3(3) clone, showed a pattern of intracellular Cx43-HKKSL localization with a more perinuclear distribution and, occasionally, Cx43-HKKSL that appeared to be localized to sites of cell-cell contact (Figure 1). Although rare, we consistently were able to generate clones with perinuclear-localized Cx43-HKKSL by transfecting HeLa cells (Das Sarma and Koval, unpublished results).

Figure 1. Differential localization of Cx43-HKKSL by a stably transfected clones. HeLa cells stably transfected with either wild type Cx43 (a) or Cx43-HKKSL (b–f) were fixed, permeabilized, and immunolabeled with anti-Cx43. Shown are five different representative HeLa/Cx43-HKKSL clones: 1(1) (b), 1(6) (c), 1(11) (d), 2(2) (e), and 3(3) (f). Most clones had Cx43-HKKSL predominantly localized to the nuclear envelope and reticular structures in the periphery of the cell, reminiscent of the ER (see Figure 2). In contrast, clone 3(3) had a large perinuclear pool of Cx43-HKKSL. 3(3) cells also showed occasional Cx43 labeling at cell-cell interfaces (arrowheads). Bar, 10 μ m.

Figure 2. Overexpressed Cx43-HKKSL colocalizes with a Golgi apparatus marker. (A) Two representative clones of HeLa cells stably transfected with Cx43-HKKSL, 1(6) (a–c) or 3(3) (d–f) were fixed, permeabilized, and immunolabeled with anti-Cx43 (a, d) and anti-calnexin (b, e) and the corresponding Cy2-labeled and Cy3-labeled fluorescent secondary antibodies. Merged images are shown in (c, f). Arrows indicate areas where Cx43-HKKSL colocalized with calnexin in 1(6) cells, indicating localization to the ER. Arrowheads indicate the perinuclear pool of Cx43-HKKSL in 3(3) cells, which does not colocalize with calnexin. (B) 1(6) (a–c) or 3(3) (d–f) cells were fixed, permeabilized and immunolabeled with anti-Cx43 (a, d) and anti-mannosidease II (mann II) (b, e) and the corresponding Cy2-labeled and Cy3-labeled fluorescent secondary antibodies. Merged images are shown in (c, f). Arrowheads show Cx43-HKKSL colocalizing with mannosidase II in both cell types. (C) 1(6) (a–c) or 3(3) (d–f) cells were fixed, permeabilized and immunolabeled with anti-Cx43 (a, d) and anti-vimentin (b, e) and the corresponding Cy2-labeled and Cy3-labeled fluorescent secondary antibodies. Merged images are shown in (c, f). Neither cell line showed significant colocalization of Cx43-HKKSL with vimentin. Bar, 10 μ m.

Immunofluorescence colocalization was done to further characterize the differences between HeLa cell transfectants with ER- vs. perinuclear-localized Cx43-HKKSL. Shown in Figure 2 are representative immunofluorescence images from the 1(6) clone of HeLa/Cx43-HKKSL, showing colocalization with an ER-marker, calnexin. In contrast to 1(6) cells, 3(3) cells showed little, if any, colocalization with calnexin (Figure 1). Instead, the 3(3) clone of HeLa/Cx43-HKKSL cells showed more colocalization with a marker for the medial Golgi apparatus, mannosidase II. Note that a portion of Cx43-HKKSL expressed by the 1(6) clone also colocalized with mannosidase II. Although these results suggest that Cx43-HKKSL is at least partly localized to an element of the Golgi apparatus, the resolution of fluorescence microscopy limits the ability to further localize this to individual Golgi stacks (17). Thus, Cx43-HKKSL expressed by the 1(6) and the 3(3) clones that colocalized with mannosidease II may be localized to different subsets of the Golgi apparatus. For instance, Cx43-HKKSL in 1(6) cells is more likely in the ER-Golgi intermediate compartment (ERGIC) (12), whereas Cx43-HKKSL in 3(3) is likely in a more distal element of the Golgi apparatus. Another possibility was that the perinuclear distribution of Cx43-HKKSL expressed by the 3(3) clone was due to aggresome formation (18). However, Cx43-HKKSL did not colocalize with vimentin, which encages aggresomes (18, 19), suggesting that Cx43-HKKSL in 3(3) cells was not misfolded.

It seemed plausible that the difference in connexin localization was due to a difference in the level of connexin expression, since overexpression can interfere with connexin assembly (20). We confirmed that this was the case by immunoblot (Figure 3), where 3(3) cells expressed 3.2 ± 0.1 (n = 3) -fold more Cx43-HKKSL than 1(6) cells. Qualitatively, clones 1(1), 1(11), and 2(2) expressed slightly more Cx43-HKKSL than 1(6) cells (Das Sarma and Koval, unpublished observations), suggesting that a threshold level of overexpression was required to permit Cx43-HKKSL to obtain a perinuclear distribution.

Figure 3. High levels of Cx43-HKKSL expression induce an intracellular Triton X-100 insoluble pool. (A) Clone 1(6) and 3(3) HeLa/Cx43-HKKSL cells were harvested and the relative total level of Cx43-HKKSL was determined by immunoblot, using β -actin as a loading control. (B) Stably transfected HeLa/Cx43-HKKSL clones were harvested, treated to separate Triton X-100 soluble and insoluble pools of Cx43-HKKSL, resolved by SDS-PAGE and analyzed by immunoblot. Shown are lanes corresponding to Triton X-100 soluble (S) and Triton X-100 insoluble (I) Cx43-HKKSL for clones 1(1), 1(6), 1(11), 2(2), and 3(3). (C) 1(6) (a, b) and 3(3) (c, d) were further characterized by in situ Triton X-100 extraction. The cells were treated for 30 min at 15°C with either control medium (a, c) or medium containing 1% Triton X-100 (b, d), then washed, fixed, permeabilized and immunostained for Cx43-HKKSL. In d, arrowheads denote Cx43-HKKSL localized to the plasma membrane, although the majority of the Triton X-100 insoluble pool was intracellular (arrows). Bar, 10 μ m.

Triton X-100 insolubility is a hallmark for gap junction formation (8, 10, 12, 21). Consistent with a lack of gap junction formation, the 1(1), 1(6), 1(11), and 2(2) clones showed virtually all of the Cx43-HKKSL in the Triton X-100 soluble pool (Figure 3). However, 3(3) cells had a significant Triton X-100 insoluble pool. In situ Triton X-100 extraction confirmed that the majority of the Triton X-100 insoluble fraction of Cx43-HKKSL corresponded to the perinuclear pool in 3(3) cells, with occasional plasma membrane labeling. Interestingly, 3(3) cells also showed low levels of Cx43-HKKSL migrating at an apparent higher Mr (6.6 ± 1.1% total (n = 3)), which was more clearly resolved by immunoblots of the Triton X-100 insoluble pool. This was most likely due to Cx43-HKKSL phosphorylation characteristic of surface localized Cx43 (7, 10, 22) and is consistent with low levels of cell surface labeling occasionally observed by immunofluorescence (e.g., Figure 1 and Figure 3). The notion that overexpressed Cx43-HKKSL has the potential to form gap junctions is further supported by a previous demonstration that HeLa cells expressing a construct with a weak—AKKFF retention signal had a combination of perinuclear and gap junction localized Cx43-AKKFF (12).

We used postembedding immunogold EM to further characterize the intracellular Triton X-100 resistant structures in 3(3) cells containing Cx43-HKKSL. As shown in Figure 4, Cx43-HKKSL formed large complexes resembling intracellular gap junctions in 3(3) cells. Structures resembling both linear arrays and annular junctions were observed, consistent with the Triton X-100 insolublility of these intracellular structures. The majority of the remaining immunogold appeared to be adjacent to membranes, however, there was occasional Cx43-HKKSL labeling that might be cytosolic instead of membrane associated (23).

Figure 4. Formation of intracellular gap junction-like structures by 3(3) cells. Clone 3(3) HeLa/Cx43-HKKSL cells were processed for postembedding immunogold EM to label Cx43-HKKSL with 20 nm gold as described in Methods. Arrows indicate annular junctions, arrowheads indicate linear gap junction like arrays. a: bar, 500 nm; b,c: bar, 100 nm.

To further characterize the Triton X-100 soluble pool of Cx43-HKKSL, sucrose gradient fractionation was used to determine the oligomerization state of Cx43-HKKSL expressed by the different clones (Figure 5). Cells which had ER-localized Cx43-HKKSL showed a sucrose gradient fractionation pattern consisting mainly of a single peak centered at ∼8–9% sucrose characteristic of monomeric Cx43-HKKSL, consistent with our previous studies (12, 13). However the 3(3) clone showed two well defined peaks of Cx43-HKKSL, corresponding to monomeric Cx43-HKKSL, centered at 8–9% sucrose, and hexameric Cx43-HKKSL, centered at ∼ 15% sucrose. Taken together, this suggests that the level of Cx43-HKKSL expression by 3(3) cells was sufficient to circumvent the normal Cx43 quality control apparatus.

Figure 5. Oligomerization of Cx43-HKKSL expressed by 3(3) cells. HeLa/Cx43-HKKSL cells were harvested, the Triton X-100 soluble fraction was prepared and analyzed by sucrose gradient fractionation as described in Methods. (A) Representative immunoblots showing the sucrose gradient fraction pattern for clone 1(6) and 3(3) HeLa/Cx43-HKKSL cells. The percent sucrose content of the fractions are denoted below each individual lane, molecular mass markers are indicated to the right. (B) Densitometric analysis of immunoblots obtained from sucrose gradient fractionated Cx43-HKKSL expressed by clones 1(1), 1(6), 1(11), 2(2) (top), and clone 3(3) cells (bottom). In contrast to the other clones, only the 3(3) clone of HeLa/Cx43-HKKSL cells showed a significant Cx43-HKKSL hexamer peak centered at ∼ 15% sucrose.

DISCUSSION

HeLa cells stably transfected to express a Cx43-HKKSL chimera containing a di-lysine ER retention/retrieval motif normally show Cx43-HKKSL present in the ER as an apparent monomer (12, 13). However, we found that overexpression of Cx43-HKKSL by HeLa cells induced Cx43-HKKSL oligomerization and formation of large Cx43-HKKSL complexes resembling intracellular gap junctions (Figure 6). Thus HeLa cells do not have an unlimited capacity to regulate Cx43-HKKSL and, by analogy, Cx43 oligomerization. Since the morphology of the ER in 3(3) cells was normal, this also suggests that Cx43-HKKSL overexpression did not interfere with the ability of the ER retention/retrieval system to operate on other ER resident proteins, such as calnexin (24). Instead, the lack of ER retrieval by clone 3(3) cells was more likely due to the formation of large complexes driven by overexpression, particularly since cells expressing lower levels readily retrieve Cx43-HKKSL that has escaped from the ER (12).

Figure 6. Models for processing Cx43-HKKSL by stably transfected HeLa cells. (a) In cells expressing low-to-moderate levels of Cx43-HKKSL, most Cx43-HKKSL is retained in the ER and ERGIC. Cx43-HKKSL which escapes into the Golgi is retrieved by the di-lysine tag. (b) When overexpressed, more Cx43-HKKSL escapes into the Golgi apparatus, where it reaches a concentration allowing oligomerization into hexamers and formation of large intracellular complexes that are resistant to retrieval. Alternatively, high levels of Cx43-HKKSL may enable early hexamer formation in the ER which, in turn, could promote efflux into the Golgi apparatus and subsequent complex formation.

Consistent with these observations, forced overexpression of transfected Cx32 by BHK cells induces the formation of intracellular complexes resembling gap junctions in the ER (20). Since wild type, overexpressed Cx32 formed intracellular gap junction like structures, this suggests that the HKKSL tag was not necessary for their formation. However, it is possible that the HKKSL tag might help promote formation of large complexes by crosslinking adjacent hexamers via coatomer interactions (25). Another possibility is that since ZO-1 is a critical cofactor to promote Cx43 trafficking to the plasma membrane and gap junction assembly (26, 27, 28) and the HKKSL tag of Cx43-HKKSL blocks the ZO-1 binding site, the inability of Cx43-HKKSL to interact with ZO-1 might contribute to the formation of large intracellular complexes.

Recently, it has also been suggested that overexpression might underlie some of the conflicting results obtained using transfected cell models to study connexin trafficking and oligomerization (29). This is a particular concern, since transfected cell models are frequently used to study trafficking and assembly defects of mutant connexins associated with human disease and suggest that care should be used in producing and interpreting these models. Another consideration when using transfected cell lines is that genomic instability can alter the expression profile of individual clones isolated from the same parental line (30). Relevant to the present study, we found that Affymetrix gene array analysis comparing 1(6) and 3(3) cells did not reveal any significant difference in gene expression between the two cell lines, suggesting that this was not the case (Das Sarma and Koval, unpublished observations).

Although connexin overexpression at the whole cell level has the potential to interfere with oligomerization, one can envision scenarios where localized increases in connexin concentration might also drive aberrant oligomerization and/or promote the formation of complexes containing multiple connexons. For instance, if a connexin mutation decreases the rate of connexin efflux from the ER this might, in turn, increase the connexin content of the early secretory pathway which, in turn, could promote early oligomerization. Consistent with this possibility, Berthoud, et al. demonstrated that the mutant form of Cx50 associated with human cataract, Cx50-P88S, forms cytoplasmic accumulations in the ER with partial plaque-like character (31). However, trafficking defects associated with mutant connexins are not necessarily associated with increased assembly, since they can also be associated with decreased assembly. For instance, two mutant Cx32 proteins associated with Charcot-Marie-Tooth disease are retained in the ER or Golgi apparatus in a monomeric form and subsequently processed by ER-associated degradation (ERAD) quality control pathway (32).

Although overexpressed Cx43-HKKSL had the capacity to form oligomers and higher order structures, it did not appear to be handled by the cells as a misfolded protein. For instance, sucrose gradient of the Triton X-100 soluble pool of Cx43-HKKSL in 3(3) cells consisted of two well-formed monomer and hexamer peaks. Also, the perinuclear Cx43-HKKSL present in 3(3) cells did not form aggresomes. Further, Cx43-HKKSL overexpression did not affect HeLa cell viability, since the cells retained high expression levels over several passages (Das Sarma and Koval, unpublished observations).

We have previously demonstrated that, in contrast to Cx43-HKKSL, Cx32-HKKSL expressed by HeLa cells oligomerizes (12, 13). Our findings that overexpression of Cx43-HKKSL can drive oligomerization suggest the possibility that our previous results with Cx32-HKKSL might be due to overexpression. However, we believe that this is not the case, since this possibility was controlled by analyzing HeLa cell transfectants where Cx32-HKKSL was ER localized and did not have a significant Triton X-100 insoluble pool (12, 13). Also, we found that a chimera containing the third transmembrane domain (TM3) and second extracellular loop domain (EL2) of Cx43 on a Cx32-HKKSL backbone (Cx32/43/32-HKKSL) was monomeric, whereas Cx32-HKKSL was not (13). Since the same epitope was used to detect both of these constructs by immunoblot, we determined that the HeLa cells were transfected to express comparable levels of Cx32/43/32-HKKSL and Cx32-HKKSL, thus ruling out a difference in expression level as the underlying cause of the difference between the oligomerization state of Cx32/43/32-HKKSL and Cx32-HKKSL. Since Cx32/43/32-HKKSL contained the minimal Cx43 motif required to prevent oligomerization of the ER retained construct, we favor a model where charged residues at the membrane/water interface of the Cx43 TM3 domain help promote a conformation that stabilizes monomeric Cx43, in contrast to the bulky hydrophobic residues present in comparable positions of the Cx32 TM3 domain that may favor oligomerization (13).

Although it is possible that oligomerization is regulated strictly by connexin concentration in the membrane, a more likely possibility is that cells have a quality control apparatus that regulates connexin oligomerization. Whether this is the case will require identifying protein components of this putative quality control pathway. HeLa cells overexpressing Cx43-HKKSL are being investigated as a system to screen for pharmacologic agents and chaperones that regulate Cx43 oligomerization. In this system, cells will be examined for treatments with the ability to alter the intracellular distribution of Cx43-HKKSL from a perinuclear to ER distribution. Although connexins typically are not stably localized to the ER, this provides a visual method for assessing changes in connexin assembly state without requiring gap junction formation. This is an advantage, since trafficking and assembly of HKKSL-tagged connexins in each individual cell is independent of the state of connexin assembly and trafficking by neighboring cells. We have found that this is particularly useful for screening cDNA libraries for potential connexin chaperone proteins, where transfection of individual clone 3(3) cells can provide a visual readout of ER localized Cx43-HKKSL (Das Sarma and Koval, unpublished results). Also, screens based on 3(3) HeLa/Cx43-HKKSL cells use the entire Cx43 protein as “bait,” including transmembrane domains and extracellular loop domains. Thus, one goal in using cells overexpressing Cx43-HKKSL for a screening approach is to provide an adjunct method to yeast two hybrid screens (33, 34), which typically require that the “bait” consists of an aqueous connexin protein fragment. Of course, as is the case with any screening method, any candidate chaperones identified using HKKSL-tagged connexins would then have to be further verified by examining their effect on untagged connexins.

ACKNOWLEDGEMENTS

Supported by NIH grants GM61012 and P01-HL019737-26, Project 3 (MK). We thank the University of Pennsylvania Biomedical Imaging Core for performing postembedding immunogold EM experiments.