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.     

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.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Different domains are critical for oligomerization compatibility of different connexins

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.     

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.     

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.     

Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43

Immunohistochemical co-localization of distinct connexins (Cxs) in junctional areas suggests the formation of heteromultimeric channels. To determine the docking effects of the heterotypic combination of Cx43 and Cx45 on the voltage-gating properties of their channels, we transfected DNA encoding Cx43 or Cx45 into N2A neuroblastoma or HeLa cells. Using a double whole-cell voltage-clamp technique, we determined macroscopic and single-channel gating properties of the intercellular channels formed. Cx43-Cx45 heterotypic channels had rectifying properties where Cx45 connexons inactivated rapidly upon hyperpolarizing voltage pulses applied to the Cx45-expressing cell. During depolarizing pulses to the Cx45-expressing cell, Cx43 connexons inactivated with substantially reduced kinetics as compared with homotypic Cx43 channels. Similar slow kinetics was observed for homotypic Cx43M257 (truncation mutant). Heterotypic channels had a main conductance whose value was predicted by the sum of corresponding homomeric connexon conductances; it was not voltage dependent and had no detectable residual conductance. The voltage-gating kinetics of heterotypic channels and their single-channel behavior implicate a role for the Cx43 carboxyl-terminal domain in the fast gating mechanism and in the establishment of residual conductance. Our results also suggest that heterotypic docking may lead to conformational changes that inhibit this action of the Cx43 carboxyl-terminal domain.     

Gap junction permeability: selectivity for anionic and cationic probes

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 ?, charge -2), carboxyfluorescein (CF; 8.2 ?; -2), and Alexa Fluor350 (AF350, 5.4 ?; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 ?, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 ?, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.     

Characterization of the Structure and Intermolecular Interactions between the Connexin40 and Connexin43 Carboxyl-terminal and Cytoplasmic Loop Domains

Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the “particle-receptor” model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.     

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha- type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian 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.     

Neonatal rat cardiomyocytes show characteristics of nonhomotypic gap junction channels

Neonatal rat cardiomyocytes mainly coexpress the connexins Cx40, Cx43, and to a small amount Cx45, leading to potential formation of mixed (heteromeric/heterotypic) gap junction channels. Using the dual-voltage clamp technique with switching clamp circuits, the authors investigated voltage sensitivity of gap junction channels between cell pairs of Cx40, Cx43, and Cx45 stably transfected HeLa cells and compared those data to data obtained from cell pairs of cultured neonatal rat cardiomyocytes. In accordance to previously published data, the relationship between normalized conductance and transjunctional voltage (g/V(j)) was quasisymmetrical for the transfected HeLa cells, indicating homotypic gap junction channels. Boltzmann curves fitted to data obtained from neonatal rat cardiomyocyte pairs expressing both Cx40 and Cx43 showed an asymmetrical inactivation pattern, which cannot be explained by the presence of pure populations of homotypic gap junction channels of either isoform. In conclusion the authors assume the additional presence of heterotypic and possibly even heteromeric gap junction channels in neonatal rat cardiomyocytes.     

Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons

Recent evidence indicatingformation of functional homomeric/heterotypic gap junction channels byconnexin40 (Cx40) and connexin43 (Cx43) raises the question of whetherdata previously interpreted as support for heteromeric channelformation by these connexins might not instead reflect the activity ofhomomeric/heterotypic channels. To address this question and to furthercharacterize the behavior of these channels, we used dual whole cellvoltage-clamp techniques to examine the junctions formed between cellsthat express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired withcells that naturally express both connexins (A7r5 cells). Rin40/Rin43cell pairs formed functional gap junctions that displayed a stronglyasymmetric voltage-dependent gating response. Single-channel eventamplitudes ranged between 34 and 150 pS, with 90- to 130-pS eventspredominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairsdemonstrating strong asymmetry. These cell pairs exhibited a variety ofsingle-channel events that were not consistent with homomeric/homotypicCx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels.These data indicate that Cx40 and Cx43 form homomeric/heterotypic aswell as heteromeric/heterotypic channels that display unique gating andconductance properties.

     

Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland

During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.     

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.     

Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.     

Cultured periodontal ligament fibroblasts express diverse connexins

Recent studies have suggested multiple functions of periodontal ligament fibroblasts (PDLFs) which may relate to the permeability of gap junctions composed of various types of connexins (Cxs). At present, 15 types of Cxs are known to exist, and six of their antibodies, anti-Cx26, Cx32, Cx37, Cx40, Cx43, and Cx45 are commercially available. This study aims to examine which types of Cxs are expressed in cultured PDLFs by an immunohistochemical method, western blotting, and RT-PCR.The study confirmed the expressions of Cx32, Cx40, Cx43, and Cx45 in PDLFs, while Cx26 and Cx37 were not detected. Considering previous reports, Cx32 may relate to the secretory function, and Cx40 and Cx45 to the contractile function of PDLFs, however, a function for Cx43 has not been specified. In the immunohistochemical examination, different localizations of Cx40/43 and Cx32/45 were established. The former were observed punctately, suggesting that a large part of Cx40/43 may exist in the cell membrane and construct gap junctions. In contrast, the latter were observed uniformly in all the cells, indicating that they are present both in the cell membrane and in the cytoplasm of the cells.     

The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities

Gap junctions have traditionally been characterized as nonspecific pores between cells passing molecules up to 1 kDa in molecular mass. Nonetheless, it has become increasingly evident that different members of the connexin (Cx) family mediate quite distinct physiological processes and are often not interchangeable. Consistent with this observation, differences in permeability to natural metabolites have been reported for different connexins, although the physical basis for selectivity has not been established. Comparative studies of different members of the connexin family have provided evidence for ionic charge selectivity, but surprisingly little is known about how connexin composition affects the size of the pore. We have employed a series of Alexa dyes, which share similar structural characteristics but range in size from molecular weight 350 to 760, to probe the permeabilities and size limits of different connexin channels expressed in Xenopus oocytes. Correlated dye transfer and electrical measurements on each cell pair, in conjunction with a three-dimensional mathematical model of dye diffusion in the oocyte system, allowed us to obtain single channel permeabilities for all three dyes in six homotypic and four heterotypic channels. Cx43 and Cx32 channels passed all three dyes with similar efficiency, whereas Cx26, Cx40, and Cx45 channels showed a significant drop-off in permeability with the largest dye. Cx37 channels only showed significant permeability for the smaller two dyes, but at two- to sixfold lower levels than other connexins tested. In the heterotypic cases studied (Cx26/Cx32 and Cx43/Cx37), permeability characteristics were found to resemble the more restrictive parental homotypic channel. The most surprising finding of the study was that the absolute permeabilities calculated for all gap junctional channels in this study are, with one exception, at least 2 orders of magnitude greater than predicted purely on the basis of hindered pore diffusion. Consequently, affinity between the probes and the pore creating an energetically favorable in-pore environment, which would elevate permeant concentration within the pore and hence the flux, is strongly implicated.     

Functional analysis of selective interactions among rodent connexins.

One consequence of the diversity in gap junction structural proteins is that cells expressing different connexins may come into contact and form intercellular channels that are mixed in connexin content. We have systematically examined the ability of adjacent cells expressing different connexins to communicate, and found that all connexins exhibit specificity in their interactions. Two extreme examples of selectivity were observed. Connexin40 (Cx40) was highly restricted in its ability to make heterotypic channels, functionally interacting with Cx37, but failing to do so when paired with Cx26, Cx32, Cx43, Cx46, and Cx50. In contrast, Cx46 interacted well with all connexins tested except Cx40. To explore the molecular basis of connexin compatibility and voltage gating, we utilized a chimera consisting of Cx32 from the N-terminus to the second transmembrane domain, fused to Cx43 from the middle cytoplasmic loop to the C-terminus. The chimeric connexin behaved like Cx43 with regard to selectivity and like Cx32 with regard to voltage dependence. Taken together, these results demonstrate that the second but not the first extracellular domain affects compatibility, whereas voltage gating is strongly influenced by sequences between the N-terminus and the second transmembrane domain.     

Connexin45 gap junction channels in rat cerebral vascular smooth muscle cells

Cells in blood vessel walls express connexin (Cx)43, Cx40, and Cx37. We recently characterized gap junction channels in rat basilar artery smooth muscle cells and found features attributable not only to these three connexins but also to an unidentified connexin, including strong voltage dependence and single channel conductance of 30-40 pS. Here, we report data consistent with identification of Cx45. Immunofluorescence using anti-human Cx45 and anti-mouse Cx45 antibodies revealed labeling between alpha-actin-positive cells, and RT-PCR of mRNA from arteries after endothelial destruction yielded amplicons exhibiting 90-98% identity with mouse Cx45 and human Cx45. Dual-perforated patch clamping was performed after exposure to oligopeptides that interfere with docking of Cx43, Cx40, or Cx45. Cell pairs pretreated with blocking peptides for Cx43 and Cx40 exhibited strongly voltage-dependent transjunctional conductances [voltage at which voltage-dependent conductance declines by one-half (V1/2) = +/-18.9 mV] and small single channel conductances (31 pS), consistent with the presence of Cx45, whereas cell pairs pretreated with blocking peptide for Cx45 exhibit weaker voltage-dependent conductances (V1/2 = +/-37.9 mV), consistent with block of Cx45. Our data suggest that Cx45 is transcribed, expressed, and forms functional gap junction channels in rat cerebral arterial smooth muscle.