Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Functional Characteristics of Heteromeric Cx40-Cx43 Gap Junction Channel Formation

Cx40:Cx43 expression ratio in A7r5 cells is augmented in growth stimulated vs. growth arrested conditions. To determine the impact of changing Cx40:Cx43 expression ratio on gap junction function, we have developed A7r5 cell lines that display Cx40:Cx43 ratios of 1:1 (6B5n) and 10:1 (A7r540C3). When Rin43 cells were paired with these coexpressing cells, there was an increasing asymmetry of voltage dependent gating as the Cx40:Cx43 ratio increased in the coexpressing cell. This asymmetry was opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In addition, when Rin43 cells were paired with coexpressing cells there was a shift toward smaller single channel event amplitudes with increasing Cx40:Cx43 ratio in the coexpressing cell. Again, this is opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In dye coupling experiments, 6B5N, A7r5, and A7r540C3 cells displayed charge and size selectivity that increased with increasing Cx40:Cx43 expression ratio. These data indicate that although the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents that comprise the channel, the dye permeability data fit what would be predicted by an increase in Cx40:Cx43 ratio.     

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.     

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.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_j and γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_j in Cx40/Cx40 pairs, but decreased g_j in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_j suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_j involved a decrease in both γ_j and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_jand γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_jin Cx40/Cx40 pairs, but decreased g_jin the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_jsuggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_jinvolved a decrease in both γ_jand Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Evidence for the Presence of a Free C-Terminal Fragment of Cx43 in Cultured Cells

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete® and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.     

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066 ± 0.005 min^− 1 (n = 65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Expression of gap junction genes during postnatal neural development

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.     

Investigation of the reciprocal relationship between the expression of two gap junction connexin proteins, connexin46 and connexin43

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.     

Heart and head defects in mice lacking pairs of connexins

Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.     

Opposing Effects of Nitric Oxide on Different Connexins Expressed in the Vascular System

Gap junctions—clusters of intercellular channels built by connexins (Cx)—are thought to be important for vascular cell functions such as differentiation, control of tone, or growth. In the vascular system, gap junctions can be formed by four different connexins (Cx37, Cx40, Cx43 and Cx45). The permeability of these connexin-formed gap junctions determines the amount of intercellular coupling and can be modulated by several vasoactive substances such as prostacyclin or nitric oxide (NO). We demonstrate here that NO has specific effects on certain connexins. Using two different techniques—injection of a fluorescent dye in single cells as well as detection of the de novoformation of gap junctions by a flow cytometry based technique—we found that NO decreases the functional coupling in Cx37 containing gap junctions whereas it increases the de novoformation of gap junctions containing Cx40. We conclude that NO, in addition to its known vasomotor effects, has a novel role in controlling intercellular coupling resulting in opposing effects depending on the specific connexin expressed in the cells.     

Cx37 and Cx43 Localize to Zona Pellucida in Mouse Ovarian Follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Functional Demonstration of Connexin—Protein Binding Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

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.     

Connexins: Sensors of epidermal integrity that are therapeutic targets

Gap junction proteins (connexins) are differentially expressed throughout the multiple layers of the epidermis. A variety of skin conditions arise with aberrant connexin expression or function and suggest that maintaining the epidermal gap junction network has many important roles in preserving epidermal integrity and homeostasis. Mutations in a number of connexins lead to epidermal dysplasias giving rise to a range of dermatological disorders of differing severity. ‘Gain of function’ mutations reveal connexin-mediated roles in calcium signalling within the epidermis. Connexins are involved in epidermal innate immunity, inflammation control and in wound repair. The therapeutic potential of targeting connexins to improve wound healing responses is now clear. This review discusses the role of connexins in epidermal integrity, and examines the emerging evidence that connexins act as epidermal sensors to a variety of mechanical, temperature, pathogen-induced and chemical stimuli. Connexins thus act as an integral component of the skin’s protective barrier.     

Multiple connexin proteins in single intercellular channels: Connexin compatibility and functional consequences

In vertebrates, the protein subunits of intercellular channels found in gap junctions are encoded by a family of genes called connexins. These channels span two plasma membranes and result from the association of two half channels, or connexons, which are hexameric assemblies of connexins. Physiological analysis of channel formation and gating has revealed unique patterns of connexin-connexin interaction, and uncovered novel functional characteristics of channels containing more than one type of connexin protein. Structure-function studies have further demonstrated that unique domains within connexins participate in the regulation of different functional properties of intercellular channels. Thus, gap junctional channels can contain more than one connexin, and this structural heterogeneity has functional consequencesin vitro. Moreover, emerging evidence for the existence of intercellular channels containing multiple connexins in native tissues suggests that the functional diversity generated by connexin-connexin interaction could contribute to complex communication patterns that have been observedin vivo.     

Functional Characterization of Oculodentodigital Dysplasia-Associated Cx43 Mutants

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.