The Inner Ear Contains Heteromeric Channels Composed of Cx26 and Cx30 and Deafness-Related Mutations in Cx26 Have a Dominant Negative Effect on Cx30

Cx26 and cx30 co-localize in tissues of the mammalian cochlea. Transfected HeLa cells were used to examine interactions between cx26 and cx30 and the effects on cx30 of four point mutations in cx26 that are associated with dominantly inherited hearing loss—W44S, G59A, D66H and R75W. When co-expressed, wtcx26 and wtcx30 trafficked to the same gap junction plaques. Cells transferred neurobiotin but not Lucifer Yellow, which passes freely through cx26 channels, suggesting cx30 affects the properties of cx26. G59A and D66H had a perinuclear localization when expressed alone but trafficked to the membrane when co-expressed with cx30. Co-expression of W44S, G59A or R75W with cx30, significantly reduced neurobiotin transfer in comparison with cells expressing cx30 only. These results indicate that cx26 and cx30 can oligomerize to form heteromeric connexons and demonstrate a dominant negative effect of some cx26 mutants on cx30. Immunogold labeling of thin sections of the cochlea showed both cx26 and cx30 distributed evenly on both sides of individual gap junction profiles. Immunoprecipitation of cochlear membrane proteins, isolated by procedures that preserve connexons, with either cx30 or cx26 antibodies precipitated both cx26 and cx30. Following co-injection of Lucifer Yellow and neurobiotin into individual supporting cells of the organ of Corti in cochlear slices, neurobiotin transferred to many cells, but Lucifer Yellow was retained in the injected cell. These observations are consistent with junctions composed of cx26/cx30 heteromeric connexons in the cochlea. The functional disruption caused by some cx26 mutations upon such heteromeric channels may underlie the non-syndromic nature of their effects on hearing.     

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.     

Correlations of Differentially Expressed Gap Junction Connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with Breast Cancer Progression and Prognosis

Background and Aims

Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.

Materials and Methods

Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.

Results

The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.

Conclusion

Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.     

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.     

Genotyping for Cx26 and Cx30 mutations in cases with congenital hearing loss

Hearing loss is the most frequent sensory defect in human being. The 13q11-q12 region contains the GJB2 and GJB6 genes, which code connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively. The 35delG, 167delT, and 235delC mutations in the Cx26 gene are the main cause for sporadic nonsyndromic hearing loss (NSHL) in many populations. The 342-kb deletion [del(GJB6-D13S1830)] of the Cx30 gene is the second most common connexin mutation after the 35delG mutation in some NSHL populations. In our study 47 hearing-impaired students were included. The Cx26 gene and the Cx30 gene were analyzed for presence of the 35delG, 167delT, and 342-kb deletion [del(GJB6-D13S1830)]. Genotyping were performed for detecting 35delG, 167delT, and del(GJB6-D13S1830) mutations using the PCR-ELISA techniques. According to the results obtained from 47 cases, the 35delG mutation was detected in 7 cases ( approximately 14.9%). Four of these mutations were determined as homozygote mutant ( approximately 8.5%), and three were determined as heterozygote mutant ( approximately 6.4%). However, 167delT and del(GJB6-D13S1830) mutations were not detected in the study group. These results support the overwhelming majority of 35delG in our study group from deafness school in our study. In conclusion, the 35delG mutation was determined as the most frequently shown mutation that leads to congenital hearing loss as in previous studies from Turkey.     

Nature of Cx30-containing channels in the adult mouse mammary gland

Oligonucleotide microarray analysis uniquely shows that several members of the connexin family of gap junction proteins are expressed by the epithelium during mouse mammary gland development. Connexin 26 (Cx26) is present throughout pregnancy and lactation, is then undetectable shortly after weaning, but reappears during involution. Additionally, Cx30 is abundant in late-pregnant and early lactating gland epithelium. From mid-pregnancy into early lactation, Cx26 and Cx30 co-localize in junctional plaques between epithelial cells, forming hemichannels of mixed connexin content. Microarray analysis also shows Cx32 is developmentally restricted to parturition, suggesting that specific modification of gap junction channel composition and/or intercellular communication pathways occurs at parturition. Specifically, heteromeric channels of all pairwise combinations are formed when these connexins are expressed within the same cells. Of these hemichannels, Cx26/Cx32 pores are increasingly sensitive to closure by taurine (an osmolyte implicated in milk protein synthesis) with increasing Cx26 content. In contrast, physiological taurine concentrations have no effect on Cx26/Cx30 and Cx30/Cx32 channel activity. Such changes in connexin expression and channel composition and their chemical modulation are discussed in relation to the various stages of mammary gland development in the adult mouse. This work was supported by grants GM36044 and GM61406 from the NIH to A.L. Harris and by generous funding from Breakthrough Breast Cancer Research to B. Gusterson.     

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.     

Cx43/beta-gal inhibits Cx43 transport in the Golgi apparatus

A connexin construct consisting of bacterial beta-galactosidase fused to the C-terminus of connexin43 (Cx43/beta-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/beta-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a sub-hexameric complex, and that Cx43/beta-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/beta-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.     

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.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts

The importance of connexins (Cxs) in cochlear functions has been demonstrated by the finding that mutations in Cx genes cause a large proportion of sensorineural hearing loss cases. However, it is still unclear how Cxs contribute to the cochlear function. Recent data (33) obtained from Cx30 knockout mice showing that a reduction of Cx diversity in assembling gap junctions is sufficient to cause deafness suggest that functional interactions of different subtypes of Cxs may be essential in normal hearing. In this work we show that the two major forms of Cxs (Cx26 and Cx30) in the cochlea have overlapping expression patterns beginning at early embryonic stages. Cx26 and Cx30 were colocalized in most gap junction plaques in the cochlea, and their coassembly was tested by coimmunoprecipitation. To compare functional differences of gap junctions with different molecular configurations, homo- and heteromeric gap junctions composed of Cx26 and/or Cx30 were reconstituted by transfections in human embryonic kidney-293 cells. The ratio imaging technique and fluorescent tracer diffusion assays were used to assess the function of reconstituted gap junctions. Our results revealed that gap junctions with different molecular configurations show differences in biochemical coupling, and that intercellular Ca²⁺ signaling across heteromeric gap junctions consisting of Cx26 and Cx30 was at least twice as fast as their homomerically assembled counterparts. Our data suggest that biochemical permeability and the dynamics of intercellular signaling through gap junction channels, in addition to gap junction-mediated intercellular ionic coupling, may be important factors to consider for studying functional roles of gap junctions in the cochlea. cochlea; coassembly; deafness     

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.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Subconductance states of Cx30 gap junction channels: data from transfected HeLa cells versus data from a mathematical model

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel. Substate conductances, gamma(j,substate), were unevenly distributed between the main-state and the residual-state conductance (gamma(j,main state) = 141 pS, gamma(j,residual state) = 21 pS). Activation of the first subgate reduced the channel conductance by approximately 30%, and activation of subsequent subgates resulted in conductance decrements of 10-15% each. Current transitions between the states were fast (<2 ms). Substate events were usually demarcated by transitions from and back to the main state; transitions among substates were rare. Hence, subgates are recruited simultaneously rather than sequentially. The incidence of substate events was larger at larger gradients of V(j). Frequency and duration of substate events increased with increasing number of synchronously activated subgates. Our mathematical model, which describes the operation of gap junction channels, was expanded to include channel substates. Based on the established V(j)-sensitivity of gamma(j,main state) and gamma(j,residual state), the simulation yielded unique functions gamma(j,substate) = f(V(j)) for each substate. Hence, the spacing of subconductance levels between the channel main state and residual state were uneven and characteristic for each V(j).     

Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties.     

Opposite Cx32 and Cx26 Voltage-Gating Response to CO2 Reflects Opposite Voltage-Gating Polarity

Previous studies have shown that the V_j-dependent gating behavior of gap junction channels is altered by CO₂ exposure. V_j-dependent channel closure is increased by CO₂ in some connexin channels and decreased in others. Since the former type of channels gate on the relatively negative side by V_j (negative gaters) and the latter at the positive side (positive gaters), it has been hypothesized that gating polarity determines the way CO₂ affects V_j closure. To test this hypothesis, we have studied the CO₂-mediated changes in V_j gating in channels made of Cx32, Cx26, or a Cx32 mutant (Cx32-N2D) in which asparagine (N) at position 2 was replaced with aspartate (D). With exposure to CO₂, Cx32 channels (negative gaters) show increased V_j-dependent closure, whereas Cx26 channels (positive gaters) respond in the opposite way to V_j. Additionally, Cx32-N2D channels (positive gaters) show decreased V_j closure with exposure to CO₂. The reciprocal Cx26 mutant, Cx26-D2N (negative gater), could not be tested because it did not express functional homotypic channels. The data support the hypothesis that polarity of fast V_j gating determines whether CO₂ increases or decreases the V_j dependent closure of gap junction channels.     

Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.     

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.

     

Alteration of Cx43:Cx40 expression ratio in A7r5 cells

Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.