Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.     

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.     

Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction.

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.     

Cytoplasmic Amino Acids within the Membrane Interface Region Influence Connexin Oligomerization

Gap junction channels composed of connexins connect cells, allowing intercellular communication. Their cellular assembly involves a unique quality-control pathway. Some connexins [including connexin43 (Cx43) and Cx46] oligomerize in the trans-Golgi network following export of stabilized monomers from the endoplasmic reticulum (ER). In contrast, other connexins (e.g., Cx32) oligomerize early in the secretory pathway. Amino acids near the cytoplasmic aspect of the third transmembrane domain have previously been shown to determine this difference in assembly sites. Here, we characterized the oligomerization of two connexins expressed prominently in the vasculature, Cx37 and Cx40, using constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) or treatment with brefeldin A to block ER vesicle trafficking. Both methods led to intracellular retention of connexins, since the cells lacked gap junction plaques. Retention of Cx40 in the ER prevented it from oligomerizing, comparable to Cx43. By contrast, ER-retained Cx37 was partially oligomerized. Replacement of two amino acids near the third transmembrane domain of Cx43 (L152 and R153) with the corresponding amino acids from Cx37 (M152 and G153) resulted in early oligomerization in the ER. Thus, residues that allow Cx37 to oligomerize early in the secretory pathway could restrict its interactions with coexpressed Cx40 or Cx43 by favoring homomeric oligomerization, providing a structural basis for cells to produce gap junction channels with different connexin composition.     

Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins.

The cellular distribution of connexin40 (Cx40), a newly cloned gap junction structural protein, was examined by immunofluorescence microscopy using two different specific anti-peptide antibodies. Cx40 was detected in the endothelium of muscular as well as elastic arteries in a punctate pattern consistent with the known distribution of gap junctions. However, it was not detected in other cells of the vascular wall. By contrast, Cx43, another connexin present in the cardiovascular system, was not detected in endothelial cells of muscular arteries but was abundant in the myocardium and aortic smooth muscle. We have tested the ability of these connexins to interact functionally. Cx40 was functionally expressed in pairs of Xenopus oocytes and induced the formation of intercellular channels with unique voltage dependence. Unexpectedly, communication did not occur when oocytes expressing Cx40 were paired with those expressing Cx43, although each could interact with a different connexin, Cx37, to form gap junction channels in paired oocytes. These findings indicate that establishment of intercellular communication can be spatially regulated by the selective expression of different connexins and suggest a mechanism that may operate to control the extent of communication between cells.     

Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals

Intercellular communication between cells of the vessel wall is established by a combination of diffusion and convection of humoral and endothelial factors in the extracellular fluid or by direct intercellular contacts present in the form of gap junctions composed of proteins called connexins. At least connexin (Cx)37, Cx40 and Cx43 are expressed in the vessel wall, but disparate findings with regard to the cell specific localisation of connexins in the vasculature indicate that the distribution of connexins may be species and vessel specific. Moreover, differences in expression exist between cells in culture and tissue sections. We performed an inventory immunohistochemical study on the localisation of Cx37, Cx40 and Cx43 on tissue sections of the bovine, micropig and rat aorta and coronary system, which represent morphologically and functionally different types of vessels in the arterial system. We could observe Cx40 labelling most commonly, although with various intensities, between endothelial and smooth muscle cells of the species studied, with the exception of rat aortic smooth muscle cells. The distribution of Cx43 is more differentiated and mostly confined to smooth muscle cells, although it can be detected scarcely between endothelial cells. Cx37, when detectable, is predominantly expressed between endothelial cells in a heterogeneous pattern. We conclude that Cx40 is the constitutive vascular gap junction protein in situ and guarantees cell coupling between cells in the vessel wall. The differentiated distribution of both Cx37 and Cx43 suggests they are involved in more dynamic processes. Accepted: 12 October 1999     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Adenoviral delivery of human connexin37 induces endothelial cell death through apoptosis

Gap junction channels formed of connexins directly link the cytoplasm of adjacent cells and have been implicated in intercellular signaling that may regulate the functions of vascular cells. To facilitate connexin manipulation and analysis of their roles in adult endothelial cells, we developed adenoviruses containing the vascular connexins (Cx37, Cx40, and Cx43). We infected cultured human umbilical vein endothelial cells with control or connexin adenoviruses. Connexin expression was verified by immunoblotting and immunofluorescence. Infection with the Cx37 adenovirus (but not control or other connexin adenoviruses) led to a dose-dependent death of the endothelial cells that was partially antagonized by the gap junction blocker alpha-glycyrrhetinic acid and altered the intercellular transfer of Lucifer yellow and neurobiotin. Cell morphology, Annexin V and TUNEL staining, and caspase 3 assays all implicated apoptosis in the cell death. These data suggest that connexin-specific alterations of intercellular communication may modulate endothelial cell growth and death.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

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.     

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.     

Levels of gap junction proteins in coronary arterioles and aorta of hamsters exposed to the cold and during hibernation and arousal.

There are marked changes in vascular dynamics during prolonged periods in the cold, entrance into hibernation, and arousal to euthermy. Cell-to-cell communication through gap junction channels plays a pivotal role in the control of vasomotor function. Multiple gap junction proteins are expressed in blood vessels, including connexins 37 (Cx37), 40 (Cx40), 43 (Cx43), and 45 (Cx45). Using immunolabeling techniques combined with confocal microscopy, we quantitated the levels of these connexins in coronary arterioles and the thoracic aorta of the golden hamster in four physiological conditions: normal control animals at euthermy; cold-exposed animals (before entrance into hibernation); during hibernation; and after 2-hr arousal from hibernation. In all groups, Cx37 was localized between endothelial cells of the aorta and Cx40 was observed between endothelial cells of coronary arterioles and the aorta. Cx43 was confined to smooth muscle cells of the aorta. Labeling for Cx45 was detected in the endothelium of the ascending aorta. The expression of Cx37 was significantly reduced in cold-exposed, hibernating, and aroused animals. Immunolabeling for Cx40 was increased in the coronary arteriolar endothelium of the cold-exposed group compared with normal controls, hibernating, and aroused animals, perhaps to facilitate intercellular communication during the prolonged circulatory changes to vascular dynamics required to maintain core temperature during cold adaptation. Cx40 expression was unchanged in the aorta. Cx43 immunoexpression in the aorta remained constant under all conditions examined. These changes in connexin expression did not occur during the rapid circulatory changes associated with arousal from hibernation.     

Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries

Connexins are the protein constituents of gap junctions which mediate intercellular communication in most tissues. In arterioles gap junctions appear to be important for conduction of vasomotor responses along the vessel. Studies of the expression pattern of connexin isoforms in the microcirculation are sparse. We investigated the expression of the three major vascular connexins in mesenteric arterioles (diameter <50 micro m) from male Sprague-Dawley rats, since conducted vasomotor responses have been described in these vessels. The findings were compared with those obtained from upstream small resistance arteries. Indirect immunofluorescence techniques were used on whole mounts of mesenteric arterioles and on frozen sections of resistance arteries (diameter approximately 300 micro m). Mesenteric arterioles expressed Cx40 and Cx43 in the endothelial layer, and Cx37 was found in most but not all vessels. Connexins were not demonstrated in the media. In resistance arteries endothelial cells expressed Cx37, Cx40 and Cx43. Ultrastructural studies of mesenteric arterioles confirmed that gap junction plaques between endothelial cells are present, whereas myoendothelial, or smooth muscle cell gap junctions could not be demonstrated. The findings suggest that smooth muscle cells in mesenteric arterioles may not be well coupled and favour that conducted vasomotor responses in these vessels are propagated through the endothelial cell layer.     

Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles

Vascular conducted responses are believed to play a central role in controlling the microcirculatory blood flow. The responses most likely spread through gap junctions in the vascular wall. At present, four different connexins (Cx) have been detected in the renal vasculature, but their role in transmission of conducted vasoconstrictor signals in the preglomerular arterioles is unknown. Connexin mimetic peptides were previously reported to target and inhibit specific connexins. We, therefore, investigated whether conducted vasoconstriction in isolated renal arterioles could be blocked by the use of mimetic peptides directed against one or more connexins. Preglomerular resistance vessels were microdissected from kidneys of Sprague-Dawley rats and loaded with fura 2. The vessels were stimulated locally by applying electrical current through a micropipette, and the conducted calcium response was measured 500 mum from the site of stimulation. Application of connexin mimetic peptides directed against Cx40, 37/43, 45, or a cocktail with equimolar amounts of each, did not inhibit the propagated response, whereas the nonselective gap junction uncoupler carbenoxolone completely abolished the propagated response. However, the connexin mimetic peptides were able to reduce dye coupling between rat aorta endothelial cells shown to express primarily Cx40. In conclusion, we did not observe any attenuating effects on conducted calcium responses in isolated rat interlobular arteries when exposed to connexin mimetic peptides directed against Cx40, 37/43, or 45. Further studies are needed to determine whether conducted vasoconstriction is mediated via previously undescribed pathways.     

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.     

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.