Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss

Connexins (Cx) form gap junctions that allow the exchange of small metabolites and ions. In the inner ear, Cx26 is the major gap junction protein and mutations in the Cx26-encoding gene, GJB2, are the most frequent cause of autosomal recessive non-syndromic hearing loss (DFNB1). We have functionally analyzed five Cx26 mutations associated with DFNB1, comprising the following single amino-acid substitutions: T8M, R143W, V153I, N206S and L214P. Coupling of cells expressing wild-type or mutant Cx26 was measured in the paired Xenopus oocyte assay. We found that the R143W, V153I and L214P mutations were unable to form functional channels. In contrast, the T8M and N206S mutants did electrically couple cells, though their voltage gating properties were different from wild-type Cx26 channels. The electrical coupling of oocytes expressing the T8M and N206S mutants suggest that these channels may retain high permeability to potassium ions. Therefore, deafness associated with Cx26 mutations may not only depend on reduced potassium re-circulation in the inner ear. Instead, abnormalities in the exchange of other metabolites through the cochlear gap junction network may also produce deafness.     

Intracellular distribution, assembly and effect of disease-associated connexin 31 mutants in HeLa cells

Mutations in connexin 31 （Cx31） are associated with erythrokeratodermia variabilis （EKV）,hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant （L34P） and four hearing impairment-associated mutants （66delD,141delI, R180X and E183K）, significantly reduced plaque formation in the cells with five EKV-associated dominant mutants （G12R, G12D, R42P, C86S and F137L） and no obvious change in the cells with two other mutants （I 141V and 652del 12）. Immunoblotting analysis showed that 12 mutated Cx31 s, like WTCx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants （G12R, G12D, R42P, C86S and F137L） caused cell death in HeLa cells. However, the five heating impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, diseaseassociated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.     

Functional studies of human skin disease- and deafness-associated connexin 30 mutations

Connexin 30 (Cx30) is a component of the gap junction complex. Dominant and recessive mutations in the GJB6 gene encoding Cx30 are associated with a variety of human inherited diseases primarily affecting the epidermis, hair, nail, and/or the inner ear. The underlying mechanism of disease associated with different GJB6 mutations such as the disruption of gap junction mediated intercellular communication is unknown. Towards understanding these disease mechanisms, transfection studies were performed in a keratinocyte cell line and in HeLa cells using EGFP tagged wildtype Cx30 and mutant Cx30 constructs harbouring dominant disease-associated GJB6 mutations. For all three of the skin disease-associated Cx30 mutations investigated, impaired trafficking of the protein to the plasma membrane was observed thus preventing the formation of functional Cx30 gap junctions. In contrast, the deafness-associated mutation T5M-Cx30/EGFP trafficked to the membrane but defective channel activity was observed following dye transfer studies.     

Exchange of serine residues 263 and 266 reduces the function of mouse gap junction protein connexin31 and exhibits a dominant-negative effect on the wild-type protein in HeLa cells

To characterize the role of Cx31 phosphorylation, serine residues 263 and 266 (Cx31Delta263,266) or 266 (Cx31Delta266) alone were exchanged for amino acids that cannot be phosphorylated. HeLa cells, which were stably transfected with wild type and the two different mutant Cx31-cDNA constructs, were analyzed for expression, phosphorylation, localization, formation of functional gap junction channels, and degradation of mutant Cx31 protein. Both mutant proteins showed similar reduced phosphorylation levels compared to Cx31 wild type, indicating a pivotal role of serine residue 266 for Cx31 phosphorylation. None of these mutations did interfere with correct intracellular trafficking of gap junction proteins. Pulse chase experiments with the different transfectants revealed an increased turnover of both mutated Cx31 proteins. They showed decreased intercellular communication as shown by dye transfer to neighboring cells and measurement of total conductance (mutant Cx31Delta263,266). Mutated Cx31 protein (Cx31Delta263,266) diminished the function of the Cx31 wild-type protein dependent on the amount of the mutated protein, indicating a dominant-negative effect of the mutated protein in HeLa cells.     

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.     

Connexin43 PDZ2 binding domain mutants create functional gap junctions and exhibit altered phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43deltaI382) or the last five amino acids (Cx43delta378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43deltaI382 and Cx43 delta378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43ΔI382) or the last five amino acids (Cx43Δ378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43ΔI382 and Cx43Δ378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.     

Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.     

Oculodentodigital dysplasia-causing connexin43 mutants are non-functional and exhibit dominant effects on wild-type connexin43

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.     

Analysis of Trafficking,Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.     

Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat mammary tumor cells

Intercellular gap junction channels are thought to form when oligomers of connexins from one cell (connexons) register and pair with connexons from a neighboring cell en route to forming tightly packed arrays (plaques). In the current study we used the rat mammary BICR-M1Rk tumor cell line to examine the trafficking, maturation, and kinetics of connexin43 (Cx43). Cx43 was conclusively shown to reside in the Golgi apparatus in addition to sites of cell-cell apposition in these cells and in normal rat kidney cells. Brefeldin A (BFA) blocked Cx43 trafficking to the surface of the mammary cells and also prevented phosphorylation of the 42-kD form of Cx43 to 44- and 46-kD species. However, phosphorylation of Cx43 occurred in the presence of BFA while it was still a resident of the ER or Golgi apparatus yielding a 43-kD form of Cx43. Moreover, the 42- and 43-kD forms of Cx43 trapped in the ER/Golgi compartment were available for gap junction assembly upon the removal of BFA. Mammary cells treated with BFA for 6 h lost preexisting gap junction "plaques," as well as the 44- and 46-kD forms of Cx43 and functional coupling. These events were reversible 1 h after the removal of BFA and not dependent on protein synthesis. In summary, we provide strong evidence that in BICR-M1Rk tumor cells: (a) Cx43 is transiently phosphorylated in the ER/Golgi apparatus, (b) Cx43 trapped in the ER/Golgi compartment is not subject to rapid degradation and is available for the assembly of new gap junction channels upon the removal of BFA, (c) the rapid turnover of gap junction plaques is correlated with the loss of the 44- and 46-kD forms of Cx43.     

Dual Functions for Connexins: Cx43 Regulates Growth Independently of Gap Junction Formation

Connexins, the family of proteins that form vertebrate gap junctions, have key roles during development and in the adult. Previously, the physiological actions of connexins have been ascribed solely to formation of gap junction channels and thought to be mediated by the transfer of small molecules between neighboring cells. In conflict with this hypothesis here we demonstrate that Cx43 can affect cell growth independently of gap junction formation. This conclusion is based on four findings: (1) There is a lack of correlation between the action of Cx43 mutants Cx43-S255A, Cx43-S279A, and Cx43-S282A on growth and cell coupling in 3T3 A31 fibroblasts. (2) Blockade of gap junction formation, by either heptan-1-ol treatment or culturing cells at low density, had no effect on the ability of the Cx43 mutants to control growth. (3) Wildtype Cx43 inhibited growth of Neuro2a cells under conditions where gap junctions were unable to form. (4) The CT domain of Cx43, which lacks intrinsic gap junction activity, is as effective as the wildtype molecule in suppressing the growth of Neuro2a cells. These observations demonstrate that Cx43 has dual functions: first, its well-accepted role in forming a gap junction channel and, second, a direct action of the connexin protein on growth that is mediated via the cytoplasmic carboxyl domain.     

Downregulation of gap junction expression and function by endoplasmic reticulum stress

Gap junctional intercellular communication (GJIC) plays a critical role in the control of multiple cell behavior as well as in the maintenance of tissue and organ homeostasis. However, mechanisms involved in the regulation of gap junctions (GJs) have not been fully understood. Given endoplasmic reticulum (ER) stress and dysfunction of GJs coexist in several pathological situations, we asked whether GJs could be regulated by ER stress. Incubation of mesangial cells with ER stress‐inducing agents (thapsigargin, tunicamycin, and AB₅ subtilase cytotoxin) resulted in a decrease in connexin 43 (Cx43) expression at both protein and mRNA levels. This was accompanied by a loss of GJIC, as evidenced by the reduced numbers of dye‐coupled cells after single cell microinjection or scrape loading dye transfer. Further studies demonstrated that ER stress significantly inhibited the promoter activity of the Cx43 gene, reduced [³⁵S]‐methionine incorporation into Cx43 protein and accelerated degradation of Cx43. ER stress also decreased the Cx43 protein levels in several different cell types, including human umbilical vein endothelial cells, mouse‐derived renin‐secreting cells and human hepatoma cells. Furthermore, induction of ER stress by hypoxic chemicals thenoyltrifluoroacetone and cobalt chloride was found to be associated with a reduction in Cx43. Our findings thus reveal a close link between ER stress and GJs. ER stress may represent a novel mechanism underlying the altered GJs in a variety of pathological situations. J. Cell. Biochem. 107: 973–983, 2009. © 2009 Wiley‐Liss, Inc.     

Atrial Fibrillation-Linked Germline GJA5/Connexin40 Mutants Showed an Increased Hemichannel Function

Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40) have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca²⁺]_o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.     

Focus on lens connexins

The lens is an avascular organ composed of an anterior epithelial cell layer and fiber cells that form the bulk of the organ. The lens expresses connexin43 (Cx43), connexin46 (Cx46) and connexin50 (Cx50). Epithelial Cx50 has critical roles in cell proliferation and differentiation, likely involving growth factor-dependent signaling pathways. Both Cx46 and Cx50 are crucial for lens transparency; mutations in their genes have been linked to congenital and age-related cataracts. Congenital cataract-associated connexin mutants can affect protein trafficking, stability and/or function, and the functional effects may differ between gap junction channels and hemichannels. Dominantly inherited cataracts may result from effects of the connexin mutant on its wild type isotype, the other co-expressed wild type connexin and/or its interaction with other cellular components.     

Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness

Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The specific role of connexin channels in the homeostasis of different organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in unaffected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identified in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identified as a polymorphism in heterozygous unaffected individuals, was devoid of function and thus may be pathologically significant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identification of functional differences of disease causing mutations may lead to define which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1.     

Evaluation of the pathogenicity of GJB3 and GJB6 variants associated with nonsyndromic hearing loss

A number of genes responsible for hearing loss are related to ion recycling and homeostasis in the inner ear. Connexins (Cx26 encoded by GJB2, Cx31 encoded by GJB3 and Cx30 encoded by GJB6) are core components of gap junctions in the inner ear. Gap junctions are intercellular communication channels and important factors that are associated with hearing loss. To date, a molecular genetics study of GJB3 and GJB6 as a causative gene for hearing loss has not been performed in Korea. This study was therefore performed to elucidate the genetic characteristics of Korean patients with nonsyndromic sensorineural hearing loss and to determine the pathological mechanism of hearing loss by analyzing the intercellular communication function of Cx30 and Cx31 variants. Sequencing analysis of the GJB3 and GJB6 genes in our population revealed a total of nine variants, including four novel variants in the two genes. Three of the novel variants (Cx31-p.V27M, Cx31-p.V43M and Cx-30-p.I248V) and two previously reported variants (Cx31-p.V84I and Cx30-p.A40V) were selected for functional studies using a pathogenicity prediction program and assessed for whether the mutations were located in a conserved region of the protein. The results of biochemical and ionic coupling tests showed that both the Cx31-p.V27M and Cx31-p.V84I variants did not function normally when each was expressed as a heterozygote with the wild-type Cx31. This study demonstrated that two variants of Cx31 were pathogenic mutations with deleterious effect. This information will be valuable in understanding the pathogenic role of GJB3 and GJB6 mutations associated with hearing loss.     

Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P₁ phosphorylated and P₀ unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.     

Dispersed and Aggregated Gap Junction Channels Identified by Immunogold Labeling of Freeze-Fractured Membranes

An indirect immunogold labeling technique was applied to replicas of freeze-fractured membranes of rapidly frozen unfixed cells. The endogenous gap junction protein Cx43 of BICR/M1R_krat mammary tumor cells was preferentially identified in quasi-crystalline gap junction plaques as were the transfected connexins Cx40, Cx43, and Cx45 in HeLa (human cervical carcinoma) cells. With this method we also detected contact areas with dispersed gap junction channels which are the only structural correlation for endogenous Cx45 in HeLa wild-type cells where no gap junction plaques exist. In double-transfected HeLa cells a colocalization of Cx40 and Cx43 was occasionally detected in quasi-crystalline gap junction plaques, whereas in contact areas with dispersed particles only one Cx type was present. Our results indicate that functional gap junction channels exist outside the quasi-crystalline plaques.