Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice

The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea.     

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     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to ~1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions

Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels.     

Connexin43: emerging role in erectile function

Connexins, that have their main function as part of gap junction channels, are proteins expressed in a large number of tissues such as endocrine, nervous, vascular, and muscular tissues. Gap junctions are implicated in tissue homeostasis and control of cell proliferation and differentiation. Interestingly, mutations of connexin genes have been reported in several human diseases (peripheral neuropathies, cardiovascular and dermatological diseases, hereditary cataract, and deafness) and altered expression of connexins have been associated with tumoral progression. Today, several lines of study argue for a critical role of gap junctions in corporal smooth muscle relaxation and erectile response. The present review highlights the emerging role of connexin43, one of these membranous proteins, in the physiology and physiopathology of human erectile function and its possible medical application.     

Inherited connexin mutations associated with hearing loss

One of the most dramatic discoveries in the field of hereditary hearing loss is the association of this sensory defect with connexin mutations. Most significant is the large proportion, 30-50%, of inherited hearing loss that is due to mutations in connexin 26. The proteins these genes encode are expressed in the cochlear duct, in regions containing gap junctions. Together, these findings suggest a crucial role for gap junction proteins in the mammalian inner ear. Mouse models with specific connexin mutations leading to deafness will help resolve the many questions regarding the role of these gap junction proteins in the inner ear.     

Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function

Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.     

Connexin mutations in hearing loss,dermatological and neurological disorders

Gap junctions are important structures in cell-to-cell communication. Connexins, the protein units of gap junctions, are involved in several human disorders. Mutations in beta-connexin genes cause hearing, dermatological and peripheral nerve disorders. Recessive mutations in the gene encoding connexin 26 (GJB2) are the most common cause of childhood-onset deafness. The combination of mutations in the GJB2 and GJB6 (Cx30) genes also cause childhood hearing impairment. Although both recessive and dominant connexin mutants are functionally impaired, dominant mutations might have in addition a dominant-negative effect on wild-type connexins. Some dominant mutations in beta-connexin genes have a pleiotropic effect at the level of the skin, the auditory system and the peripheral nerves. Understanding the genotype-phenotype correlations in diseases caused by mutations in connexin genes might provide important insight into the mechanisms that lead to these disorders.     

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H₂S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H₂S.     

Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.     

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.     

Developmental expression patterns of connexin26 and -30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre- and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and -30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22-week-old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear.     

缝隙连接及其与人类疾病的关系

缝隙连接是由多基因家族编码的连接蛋白构成的、细胞间的跨膜水相通道。目前已确定小鼠连接蛋白基因家族含有20个成员,人类连接蛋白基因家族含有21个成员,其中有19种在人类和小鼠中均有表达,具有很高的同源性;不同的连接蛋白可形成同型和异型两种连接子,不同类型连接子可形成4种不同类型的缝隙连接通道。越来越多的研究表明,连接蛋白基因突变与人类遗传性疾病密切相关。     

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus)

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.     

Relevance of connexin deafness (DFNB1) to human evolution

The connexins are the subunits of a family of proteins that form gap junctions, allowing ions and small molecules to move between adjacent cells. At least four connexins are expressed in the ear, and, although there are known mutations at >100 loci that can cause deafness, those involving DFNB1, in the interval 13q11-q12 containing the GJB2 and GJB6 genes coding for connexins 26 and 30, are the most frequent cause of recessive deafness in many populations. We have suggested that the combined effects of relaxed selection and linguistic homogamy can explain the high frequency of connexin deafness and may have doubled its incidence in this country during the past 200 years. In this report, we show by computer simulation that assortative mating, in fact, can accelerate dramatically the genetic response to relaxed selection. Along with the effects of gene drift and consanguinity, assortative mating also may have played a key role in the joint evolution and accelerated fixation of genes for speech after they first appeared in Homo sapiens 100,000-150,000 years ago.     

Gap junction remodeling and cardiac arrhythmogenesis: cause or coincidence?

Gap junctions, clusters of transmembrane channels that link adjoining cells, mediate myocyte-to-myocyte electrical coupling and communication. The component proteins of gap junction channels are termed connexins and, in in vitro expression systems, gap-junctional channels composed of different connexin types exhibit different biophysical properties. In common with other tissues, the heart expresses multiple connexin isoforms. Spatially defined patterns of expression of three connexin isoforms - connexin43, connexin40 and connexin45 - form the cell-to-cell conduction pathways responsible for the orderly spread of current flow that governs the normal cardiac rhythm. Remodeling of gap junction organization and connexin expression is a common feature of human heart disease conditions in which there is an arrhythmic tendency. This remodeling may take the form of disturbances in the distribution of gap junctions and/or quantitative alterations in connexin expression, notably reduced ventricular connexin43 levels. The idea that such changes may contribute to the development of a pro-arrhythmic substrate in the diseased heart has gained ground over the last decade. Recent studies using transgenic mice models have raised new opportunities to explore the significance of gap junction remodeling in the diseased heart.     

Emerging role of gap junctions in epilepsy

This review highlights the contribution of gap junctions to the pathophysiology of epilepsy. The tissue expression and spatiotemporal regulation of connexins is discussed, and the phenotypes of specific connexin knockouts are considered. Electrophysiologic studies have implicated gap junctions in the generation of very fast oscillations preceding seizures. Gap junction inhibitors have shown powerful anticonvulsant effects, to date primarily in in vitro studies. Specific inhibition of gap junctions in vivo along with more detailed human tissue studies are needed to understand more fully the role of gap junctions in epileptogenesis.