Gap Junction Plaques Research Articles

Redistribution of connexin 43 during cell division.

Gap junction channels, once clustered into gap junction plaques, allow communication of essential metabolites between cells. Gap junction plaques have been reported to be lost from the cell surface during cell division. The mechanism involved in this loss of gap junction plaques during mitosis is unclear, but we hypothesize that an endoexocytotic mechanism that results in cytoplasmic double-membraned annular gap junction vesicles is involved. In this study, gap junction plaque changes in dividing cells were examined in SW-13 adrenocortical tumor cells. Endogenous gap junction protein, connexin 43 (Cx43), was detected with immunofluorescence, and live cell imaging was used to monitor green fluorescent protein-tagged Cx43 (Cx43-GFP). Mitotic stages were identified by Hoechst chromosomal staining. During interphase, large gap junction plaques were detected; however, the presence of these plaques decreased, whereas cytoplasmic puncta increased beginning with prophase. The cytoplasmic puncta were demonstrated with immunoelectron microscopy to be Cx43- positive annular gap junction vesicles. As gap junction plaques reformed at cleavage furrows between daughter cells, the number of annular gap junctions decreased during cytokinesis. The data are consistent with the mechanism of gap junction plaque loss during mitosis relying on an endoexocytotic process that results in annular gap junction vesicles formation. The rapid formation of gap junction plaques during cytokinesis points to the intriguing possibility of connexin recycling from annular gap junction vesicles to form gap junction plaques as mitosis is completed.

Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.

The E368Q Mutant Allele of GJA8 is Associated with Congenital Cataracts with Intrafamilial Variation in a South Indian Family

To determine the basis of the autosomal dominant congenital cataracts in a three generation south Indian pedigree. The proband and several family members underwent a complete ophthalmic examination. The coding regions of eight candidate genes (CRYAA, CRYBB2, CRYGC, CRYGD, GJA3, GJA8, AQP0, and PITX3) were amplified by PCR and directly sequenced. Wild type and mutant connexin50 (Cx50) were expressed by stable transfection of HeLa cells. Their cellular distributions and function were examined by immunofluorescence microscopy and by microinjection of gap junction permeant tracers, respectively. Congenital cataracts (with some variations in phenotype) segregated as an autosomal dominant trait within a three generation pedigree. Three affected individuals (proband, sibling and mother) showed a sequence variation in the candidate gene GJA8 encoding Cx50: a c.1102G>C transversion encoding a substitution of glutamate for glutamine at position 368 (E368Q). This substitution was absent from an unaffected family member (paternal aunt) and 100 healthy controls of the same ethnicity. In transfected HeLa cells, both wild type Cx50 and E368Q localized to gap junction plaques, and supported similar levels of intercellular transfer of Neurobiotin. The E368Q mutant allele of GJA8 is associated with autosomal dominant congenital cataracts with phenotypic variability. E368Q forms gap junction plaques and functional channels in transfected HeLa cells.

Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo.

Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition to gap junction communication in primary astrocytes. After infection, Cx43 did not traffic normally to the membrane to form gap junction plaques, and that could be the basis of reduced functional gap junction coupling between astrocytes. This is an important first step toward understanding how viruses affect Cx43 expression and trafficking at the cellular level. This may provide a basis for understanding how structural alterations of astrocytic gap junctions can disrupt gap junction communication between other CNS cells in altered CNS environments due to infection and inflammation. More specifically, alteration of Cx43 may be the basis of the destabilization of Cx47 in oligodendrocytes seen in and around inflammatory demyelinating plaques in MS patients.

CRTC2 and Nedd4 ligase involvement in FSH and TGFβ1 upregulation of connexin43 gap junction.

The major mission of the ovarian follicle is the timely production of the mature fertilizable oocyte, and this is achieved by gonadotropin-regulated, gap junction-mediated cell-cell communication between the oocyte and surrounding nurturing granulosa cells. We have demonstrated that FSH and transforming growth factor beta 1 (TGFβ1) stimulate Gja1 gene-encoded connexin43 (Cx43) gap junction formation/function in rat ovarian granulosa cells is important for their induction of steroidogenesis; additionally, cAMP-protein kinase A (PKA)- and calcium-calcineurin-sensitive cAMP response element-binding (CREB) coactivator CRTC2 plays a crucial role during steroidogenesis. This study was to explore the potential molecular mechanism whereby FSH and TGFβ1 regulate Cx43 synthesis and degradation, particularly the involvement of CRTC2 and ubiquitin ligase Nedd4. Primary culture of granulosa cells from ovarian antral follicles of gonadotropin-primed immature rats was used. At 48 h post-treatment, FSH plus TGFβ1 increased Cx43 level and gap junction function in a PKA- and calcineurin-dependent manner, and TGFβ1 acting through its type I receptor modulated FSH action. Chromatin-immunoprecipitation analysis reveals FSH induced an early-phase (45 min) and FSH+TGFβ1 further elicited a late-phase (24 h) increase in CRTC2, CREB and CBP binding to the Gja1 promoter. Additionally, FSH+TGFβ1 increased the half-life of hyper-phosphorylated Cx43 (Cx43-P2). Also, the proteasome inhibitor MG132 prevented the brefeldin A (blocker of protein transport through Golgi)-reduced Cx43-P2 level and membrane Cx43 gap junction plaque. This is associated with FSH+TGFβ1-attenuated Cx43 interaction with Nedd4 and Cx43 ubiquitination. In all, this study uncovers that FSH and TGFβ1 upregulation of Cx43 gap junctions in ovarian granulosa cells critically involves enhancing CRTC2/CREB/CBP-mediated Cx43 expression and attenuating ubiquitin ligase Nedd4-mediated proteosomal degradation of Cx43 protein.

The cataract related mutation N188T in human connexin46 (hCx46) revealed a critical role for residue N188 in the docking process of gap junction channels

The mutation N188T in human connexin46 (hCx46) correlates with a congenital nuclear pulverulent cataract. This mutation is in the second extracellular loop, a domain involved in docking of gap junction hemichannels. To analyze the functional consequences of this mutation, we expressed hCx46N188T and the wild type (hCx46wt) in Xenopus oocytes and HeLa cells. In Xenopus oocytes, hemichannels formed by hCx46wt and hCx46N188T had similar electrical properties. Additionally, a Ca2+ and La3+ sensitive current was observed in HeLa cells expressing eGFP-labeled hCx46wt or eGFP-labeled hCx46N188T. These results suggest that the N188T mutation did not alter apparent expression and the membrane targeting of the protein. Cells expressing hCx46wt-eGFP formed gap junction plaques, but plaques formed by hCx46N188T were extremely rare. A reduced plaque formation was also found in cells cotransfected with hCx46N188T-eGFP and mCherry-labeled hCx46wt as well as in cocultured cells expressing hCx46N188T-eGFP and hCx46wt-mCherry. Dye transfer experiments in cells expressing hCx46N188T revealed a lower transfer rate than cells expressing hCx46wt. We postulate that the N188T mutation affects intercellular connexon docking. This hypothesis is supported by molecular modeling of hCx46 using the crystal structure of hCx26 as a template. The model indicated that N188 is important for hemichannel docking through formation of hydrogen bonds with the residues R180, T189 and D191 of the opposing hCx46. The results suggest that the N188T mutation hinders the docking of the connexons to form gap junction channels. Moreover, the finding that a glutamine substitution (hCx46N188Q) could not rescue the docking emphasizes the specific role of N188.

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.

New cellular mechanisms of gap junction degradation and recycling.

Connexins (Cxs), the constitutive proteins of gap junctions, are key actors of many physiological processes. Therefore, alterations of Cx expression and degradation lead to the development of physiopathological disorders. Because of the formation of a double membrane vesicle termed annular gap junction (AGJ), gap junction degradation is a unique physiological process for which many cellular aspects remain unclear. By using a combination of time-lapse fluorescence microscopy and high-resolution transmission electron microscopy, we evidenced new specific cellular events concerning gap junction degradation and recycling. Indeed, by time lapse video microscopy we demonstrated, for the first time to our knowledge, that an entire AGJ can be fully recycled back to the plasma membrane. Moreover, we dissected the degradative processes of gap junction by electron microscopy approaches. Interestingly, in addition to canonical autophagy and heterophagy pathways, previously described, we discovered that both pathways could sometimes intermingle. Strikingly, our results also highlighted a new lysosome-based autophagy pathway that could play a pivotal role in common autophagy degradation. The present investigation reveals that AGJ degradation is a more complex process that it was previously thought. First, a complete recycling of the gap junction plaque after its internalisation could occur. Second, the degradation of this peculiar double membrane structure is possible through autophagy, heterophagy, hetero-autophagy or by lysosomal-based autophagy. Altogether, this work underlines novel aspects of gap junction degradation that could be extended to other cell biology processes.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study.

It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated “ephaptic” mechanisms, facilitating propagation of action potentials in tandem with direct GJ-mediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus and was associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1675-z) contains supplementary material, which is available to authorized users.

Self-Structuring of Motile Astrocytic Processes within the Network of a Single Astrocyte

Dynamic structuring and functions of perisynaptic astrocytic processes and of the gap junction network within a single astrocyte are outlined. Motile perisynaptic astrocytic processes are generating microdomains. By contacting and retracting of their endfeet an appropriate receptor pattern is selected that modulates the astrocytic receptor sheath for its activation by neurotransmitter substances, ions, transporters, etc. This synaptic information processing occurs in three distinct time scales of milliseconds to seconds, seconds to minutes, hours or longer. Simultaneously, the interconnecting gap junctions are activated by building a network within the astrocyte. Frequently activated gap junction cycles become embodied in gap junction plaques. The gap junction network formation and gap junction plaques are governed and controlled in the same time scales as synaptic information processing. Biomimetic computer systems may represent an alternative to limitations of brainphysiological research. The model proposed allows the interpretation of affective psychoses and schizophrenia as time disorders basically determined by a shortened, prolonged or lacking time scale of synaptic information processing.

Effects of the Connexin43 CT on Gap Junction Sub-Domain Organization and Myocyte-Fibroblast Interactions in the Injury Border Zone

Phosphorylation of connexin43 (Cx43) has been shown to regulate gap junction (GJ) intercellular communication. A Cx43 carboxyl-terminus(CT) mimetic peptide (aCT1) increases GJ size via reduced Cx43/ZO-1 interaction in vitro and increases phosphorylation of Cx43 at aa S368 (Cx43pS368) in vitro and in vivo. We previously reported the effects of aCT1-treatment in a left ventricular (LV) cryoinjury model in vivo (Circ Res 2011;108(6):704-15). At 8-weeks after cryoinjury, histological assessment showed decreases in scar size and increases in collagen fibril uniformity in aCT1-treated hearts versus controls. Interdigitation of collagen and fibroblasts between myocytes in the injury border zone (IBZ) was reduced in treated hearts versus controls. Treated hearts exhibited a decreased propensity for induced arrhythmias (p<0.02) and an increase in Cx43pS368 in the IBZ which correlated with a tendency for Cx43 to be maintained at intercalated discs.To study the mechanistic effect of aCT1 on myocyte-fibroblast interactions via Cx43, we created a 3D heterocellular system to model the injury border zone (IBZ) in vitro. Neonatal rat ventricular fibroblasts were seeded to form a shell around a core of aggregated neonatal rat ventricular myocytes (NRVMs) in 96-well micromolds. After 72 hours, aCT1-treatment of aggregates induced an increase in heterocellular interactions via total Cx43 (p=0.03) and Cx43pS368 (p=0.01). Studies of an in vivo LV cryoinjury model were used to validate these data. Changes in myocyte-fibroblast interactions, GJ plaque size, and GJ sub-domain organization after aCT1 treatment were detected with confocal and super-resolution microscopy (both gSTED and STORM). Super-resolution microscopy enabled resolution of discrete phospho-S368 non-phosphorylated subdomains of Cx43 within GJ plaques. In sum, our results indicate the potential for Cx43 CT effects on heterocellular interactions via Cx43 in the IBZ and scar following myocardial infarction.

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions

In the present study we investigated the life cycle, trafficking, assembly and cell surface dynamics of a poorly characterized connexin family member, connexin 30 (Cx30; also known as GJB6), which plays a critical role in skin health and hearing. Unexpectedly, Cx30 localization at the cell surface and gap junctional intercellular communication was not affected by prolonged treatments with the endoplasmic reticulum (ER)-Golgi transport inhibitor brefeldin A or the protein synthesis inhibitor cycloheximide, whereas Cx43 (also known as GJA1) was rapidly cleared. Fluorescent recovery after photobleaching revealed that Cx30 plaques were rebuilt from the outer edges in keeping with older channels residing in the inner core of the plaque. Expression of a dominant-negative form of Sar1 GTPase led to the accumulation of Cx30 within the ER, in contrast to a report that Cx30 traffics via a Golgi-independent pathway. Co-expression of Cx30 with Cx43 revealed that these connexins segregate into distinct domains within common gap junction plaques, suggesting that their assembly is governed by different mechanisms. In summary, Cx30 was found to be an unusually stable, long-lived connexin (half-life >12 h), which may underlie its specific role in the epidermis and cochlea.

Gap junctional coupling is essential for epithelial repair in the avian cochlea.

The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration.

The connexin46 mutant, Cx46T19M, causes loss of gap junction function and alters hemi-channel gating.

An N-terminal mutant of connexin46 (T19M) alters a highly conserved threonine and has been linked to autosomal dominant cataracts. To study the cellular and functional consequences of substitution of this amino acid, T19M was expressed in Xenopus oocytes and in HeLa cells. Unlike wild-type Cx46, T19M did not induce intercellular conductances in Xenopus oocytes. In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques. Expression of rat T19M was cytotoxic, as evidenced by an almost complete loss of viable cells expressing the mutant protein by 48–72 h following transfection. When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity. Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca2+. Whole cell patch clamp studies of single transfected HeLa cells demonstrated that rat T19M formed functional hemi-channels with altered voltage-dependent gating. These data suggest that T19M causes cataracts by loss of gap junctional channel function and abnormally increased hemi-channel activity. Furthermore, they implicate this conserved threonine in both gap junction plaque formation and channel/hemi-channel gating in Cx46.Electronic supplementary materialThe online version of this article (doi:10.1007/s00232-014-9752-y) contains supplementary material, which is available to authorized users.

Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

BackgroundInositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.ResultsIn neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.ConclusionsThese results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0058-6) contains supplementary material, which is available to authorized users.

Deficiency of transcription factor Brn4 disrupts cochlear gap junction plaques in a model of DFN3 non-syndromic deafness.

Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome–linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.

Exome sequencing reveals mutation in GJA1 as a cause of keratoderma-hypotrichosis-leukonychia totalis syndrome.

Keratoderma-hypotrichosis-leukonychia totalis syndrome (KHLS) is an extremely rare, autosomal-dominant disorder characterized by severe skin hyperkeratosis, congenital alopecia and leukonychia totalis. The genetic defect underlying KHLS remained undetermined. By performing whole-exome sequencing in a family with KHLS, we identified a heterozygous mutation (c.23G>T [p.Gly8Val]) in GJA1, which cosegregated with the phenotype in the family. In an additional affected individual, we also found the identical de novo mutation which was absent in his unaffected family members. GJA1 encodes a gap junction protein connexin 43 (Cx43) which is ubiquitously expressed in various organs, including the epidermis and hair follicles. In vitro studies on HEK293 cells expressing Cx43(Gly8Val) found that the protein formed gap junction plaques between adjacent transfected cells, as observed in the wild-type. Dye-transfer experiments by microinjection of Lucifer yellow displayed functional gap junction of the Cx43(Gly8Val) mutant. Using patch clamp and Ca(2+) imaging methods, we observed that the Cx43(Gly8Val) hemichannel had significantly more openings than Cx43(WT), facilitating Ca(2+) influx at resting potential. Such gain-of-function effect might result in cytoplasmic Ca(2+) overload, accelerated apoptosis of keratinocytes and subsequent skin hyperkeratosis. Taken together, our results demonstrated that, with probably enhanced hemichannel activities, a mutation in GJA1 is linked to KHLS without extracutaneous involvement.

Electron microscopy and functional analysis of recombinant innexin gap junctions

Innexin is a molecular component of invertebrate gap junctions, which have an important role in neural and muscular electrical activity in invertebrates. Although the structure of vertebrate connexin26 was revealed by X-ray crystallography [1], the structure of innexin channels remains poorly understood. To study the structure of innexin gap junction channels, we expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels, and characterized their molecular dimensions and channel permeability using electron microscopy (EM) and a fluorescent dye transfer assay, respectively [2]. Negative-staining and thin-section EM of isolated INX-6 gap junction plaques revealed a loosely packed hexagonal lattice. We performed single particle analysis of purified INX-6 channels with negative-staining and cryo EM. Based on the negative-stain EM images, the class average of the junction form had a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class average of the hemichannels had diameters of up to 140 Å in the presence of detergent micelles. Cryo EM images revealed rotational peaks that could be related to the INX-6 subunits. Structural analysis of the reconstituted INX-6 channels with single particle analysis and electron tomography suggested that the oligomeric number of the INX-6 channel was distinct from that of the dodecameric connexin channel. Dye transfer experiments indicated that the INX-6-GFP-His channels were permeable to 3-kDa and 10-kDa dextran-conjugated tracers. These findings indicate that INX-6 channels have a characteristic oligomer component that differs from that in connexin gap junction channels.

AMSH‐mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions

Gap junctions (GJs) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct intercellular communication between eukaryotic cells. Although Cx43 has long been known to be a substrate for ubiquitination, the reversal of this modification by deubiquitylases (DUBs) has never been described. Here we report that the DUB-associated molecule with the SH3 domain of STAM (AMSH) interacts with Cx43 and mediates its deubiquitination. In this study, we demonstrate that Cx43 is modified with lysine 63-linked polyubiquitin chains and that these increase the interaction between Cx43 and AMSH. We also show that AMSH is recruited to GJ plaque sites at the plasma membrane, where it mediates the deubiquitination of Cx43. Using siRNA depletion or overexpression of a catalytically inactive mutant of AMSH, we show that by decreasing Cx43 deubiquitination, both the internalization and degradation rate of Cx43 are increased. Overall, these data strongly suggest that AMSH-mediated deubiquitination of Cx43 protects GJs from degradation.