Gap junction remodeling and altered connexin43 expression in the failing human heart

Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analyis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies.     

Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells

Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased ³²P phosphate incorporation into the Cx45 protein in transfected cells.The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and –26 were coexpressed in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.     

Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Connexin Expression and Cell Coupling Fail to Reverse the v-src Transformed Growth Characteristics of a Cx43-/- Cell

Gap junctions, composed of connexins, have been shown to suppress transformation in a variety of malignancies and transformed cell types. In addition, transforming factors such as the src oncogene have been shown to directly phosphorylate some connexins (e.g., Cx43) and inhibit coupling. To investigate the role of gap junctions in cell transformsation by v-src, we utilized a clonal cell line derived from Cx43 knockout mice (KoA) that was immortalized, but not transformed. Transfection by v-src induced a marked transformed phenotype characterized by growth in low serum and anchorage-independent conditions. Subsequent transfections by Cx43, Cx32 or vector alone were then tested for their effects on growth. Activity of pp60^{v - src} was confirmed in all transfectants as well as the ability of pp60^{v - src} to phosphorylate Cx43 in several clones. Despite the documented effect of pp60^{v - src} on Cx43 channel closure, modest coupling was still retained in many of the Cx43 and Cx32 transfectants. However, none of the four Cx43 transfected clones showed significant inhibitory effects on proliferation in either anchorage-independent or low serum growth conditions. Of the Cx32 clones, only one in five showed effects on growth in both assays, which was the same ratio observed for the control transfectants. Thus, based on the levels of expression achieved, which were comparable to endogenous levels in established cell lines, neither Cx43 nor Cx32 serve as effective suppressors of the transformed growth phenotype of this v-src expressing cell line.     

Regulation of connexin biosynthesis, assembly, gap junction formation, and removal

Gap junctions (GJs) are the only known cellular structures that allow a direct transfer of signaling molecules from cell-to-cell by forming hydrophilic channels that bridge the opposing membranes of neighboring cells. The crucial role of GJ-mediated intercellular communication (GJIC) for coordination of development, tissue function, and cell homeostasis is now well documented. In addition, recent findings have fueled the novel concepts that connexins, although redundant, have unique and specific functions, that GJIC may play a significant role in unstable, transient cell-cell contacts, and that GJ hemi-channels by themselves may function in intra-/extracellular signaling. Assembly of these channels is a complicated, highly regulated process that includes biosynthesis of the connexin subunit proteins on endoplasmic reticulum membranes, oligomerization of compatible subunits into hexameric hemi-channels (connexons), delivery of the connexons to the plasma membrane, head-on docking of compatible connexons in the extracellular space at distinct locations, arrangement of channels into dynamic, spatially and temporally organized GJ channel aggregates (so-called plaques), and coordinated removal of channels into the cytoplasm followed by their degradation. Here we review the current knowledge of the processes that lead to GJ biosynthesis and degradation, draw comparisons to other membrane proteins, highlight novel findings, point out contradictory observations, and provide some provocative suggestive solutions.     

Cataract-linked serine mutations in the gap junction protein connexin50 expose a sorting signal that promotes its lysosomal degradation

Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts.     

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein,in mammal heart and brain

Summary According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314–322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrathin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions.The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43.Proteins ofM _r 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

The carboxy‐terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C‐tail) to demonstrate that the C‐tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C‐tail in mediating Cx43‐dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1–242) and a construct encompassing only the C‐tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C‐tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C‐tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time‐lapse imaging to examine the behavior of migrating cells and it was apparent that the C‐tail was associated with a lamellipodia‐based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C‐tail is sufficient to induce Cx43‐dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton. J. Cell. Biochem. 110: 589–597, 2010. © 2010 Wiley‐Liss, Inc.     

The connexin43 gap junction protein is phosphorylated by protein kinase A and protein kinase C: In vivo and in vitro studies

There is general agreement that the connexin43 gap junction protein is a substrate for phosphorylation by protein kinase C but there is no similar consensus regarding the action of protein kinase A. Our previous studies demonstrated that channels formed by connexin43 were reversibly gated in response to microinjected protein kinase A and protein kinase C, but we did not determine whether these effects involved direct action on the connexin43 protein. Using a combination of in vivo metabolic labeling and in vitro phosphorylation of recombinant protein and synthetic peptides, we now find that connexin43 is a relatively poor substrate for purified protein kinase A compared to protein kinase C, but that phosphorylation can be accelerated by 8-Br-cAMP (8-bromoadenosine 3,5-cyclic monophosphate) which also enhances connexin43 synthesis but at a much slower rate than phosphorylation. Phosphorylation of a critical amino acid, Ser364, by protein kinase A, appears to be necessary for subsequent multiple phosphorylations by protein kinase C. However, protein kinase C can phosphorylate connexin43 at a reduced level in the absence of prior phosphorylation. The results suggest that the correct regulation of channels formed by connexin43 may require sequential phosphorylations of this protein by protein kinase A and protein kinase C.     

Gap junction blockage limits intercellular spreading of astrocytic apoptosis induced by metabolic depression

Astrocytes are highly coupled by gap junction channels, which allow transfer of intracellular signalling molecules and metabolites between connected cells. Astrocytic gap junctions remain open during ischemic conditions as previously demonstrated in vitro and in situ. In this study, we investigated the effect of gap junction blockage on iodoacetate-induced ATP depression and cell death progression in astrocytes in primary rat hippocampal cultures. We demonstrated that blockage of gap junctions during iodoacetate-induced inhibition of the glycolysis induced an earlier onset of the ATP depression. Moreover, initiation of apoptotic processes, demonstrated by binding of Annexin V, was critically dependent on the ATP levels. The apoptotic event was also shown to spread and involve neighbouring cells, a process that was inhibited by blockage of gap junction communication. Chelating intracellular calcium using BAPTA-AM decelerated the iodoacetate-induced ATP depression. The chelation also decelerated the spreading of apoptotic processes. Inhibition of caspases did not alter the expansion of cell groups being Annexin V positive. However, the proportion of Annexin V positive cells also being propidium iodide positive was increased after caspase inhibition. The results show that inhibition of gap junctions during cellular metabolic depression interferes with the metabolic status and cell death progression in astrocytes.     

Degradation of Connexin43 Gap Junctions Involves both the Proteasome and the Lysosome

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1–3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin–proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.     

Heteromeric connexons formed by the lens connexins, connexin43 and connexin56

In the eye lens, three connexins have been detected in epithelial cells and bow region/differentiating fiber cells, suggesting the possible formation of heteromeric gap junction channels. To study possible interactions between Cx56 and Cx43, we stably transfected a normal rat kidney cell line (NRK) that expresses Cx43 with Cx56 (NRK-Cx56). Similar to the lens, several bands of Cx56 corresponding to phosphorylated forms were detected by immunoblotting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization of Cx56 with Cx43 in the perinuclear region and at appositional membranes. Connexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstrated by sedimentation through sucrose gradients. Immunoprecipitation of Cx56 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions between the two connexins. Double whole-cell patch-clamp experiments showed that the voltage-dependence of Gmin in NRK-Cx56 cells differed from that in NRK cells. Moreover, stable interactions between Cx43 and Cx56 were also demonstrated in the embryonic chicken lens by co-precipitation of Cx43 in Cx56 immunoprecipitates. These data suggest that Cx43 and Cx56 form heteromeric connexons in NRK-Cx56 cells as well as in the lens in vivo leading to differences in channel properties which might contribute to the variations in gap junctional intercellular communication observed in different regions of the lens.     

Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43

Using an established gap junction (GJ) assembly system with experimentally reaggregated cells, we analyzed "formation plaques" (FPs), apparent sites of GJ assembly. Employing freeze-fracture electron microscopy methods combined with filipin labeling of sterols and immunolabeling for connexin43 (Cx43), we demonstrated that FPs constitute distinct membrane "domains" and that their characteristic 10-nm particles contain connexin43, thus representing precursors (i.e., GJ hemichannels) engaged in assembly. Analysis of FPs in new systems-HeLa and N2A cells-resolved questions surrounding several key but poorly understood steps in assembly, including matching of FP membranes in apposed cells, reduction in the separation between FP membranes during assembly, and the process of particle aggregation. Findings also indicated that "docking" of GJ hemichannels occurs within FP domains and contributes to reduction of intermembrane separation between FPs. Other experiments demonstrated that FPs develop following a major C-terminal truncation of Cx43 (M257), although assembly was delayed. Particle aggregation also occurred at lower densities, and densities of particles within developing GJ aggregates failed to achieve full-length levels. With regard to regulation, inhibition of assembly following protein kinase C activation failed to occur in the M257 truncation mutants, as measured by intercellular dye transfer. However, several C-terminal serine mutations failed to disrupt inhibition.     

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic G_j-V_j curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.