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The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which—together with computational studies—elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural–functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects. Cryo-electron microscopy structures of connexin channels composed of connexin 46 and connexin 50 in an open-state reveal features that govern permselectivity and the location of mutated residues linked to herediatry cataracts.

Connexin 26 (Cx26), a gap junction protein, is poorly understood in the context of cardiac milieu, including extracellular vesicles (EVs). Here, we report for the first time the presence of Cx26 on EVs obtained from human induced pluripotent stem cell-derived cardiomyocytes and human plasma. Using an in vitro model of oxidative stress and apoptosis in dH9c2 cardiomyocytes, we observed a significant decrease in Cx26 levels in EVs released by injured cells, accompanied by changes in EV concentration, particularly in exosomes. Our findings revealed that Cx26 modulates the selective loading of specific microRNAs, namely miR-1 and miR-30a, into EVs, suggesting a novel non-canonical, gap junction-independent role of Cx26 in EV-mediated cardiac signaling. Analysis of plasma EVs from healthy donors confirmed the presence of Cx26-positive EVs of cardiomyocyte origin, indicated by co-staining with cardiac troponin T. These findings suggest that further studies on the measurement of Cx26 on circulating EVs from patients with ischemic heart disease and heart failure are warranted to clarify its potential as a biomarker for cardiomyocyte injury in cardiomyopathies with oxidative stress and apoptosis.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

HIV-1 primarily targets two groups of cells in vivo : CD4 + T lymphocytes and myeloid lineage cells, such as macrophages and dendritic cells. Although myeloid cells are more resistant to HIV-1 infection than CD4 + T cells, some cytokines, including interleukin (IL)-4 and IL-6, promote myeloid cell infection. Gap junction protein beta 2 (GJB2) is particularly relevant in the field of auditory science. Here, we identified GJB2 as a novel antiviral factor by demonstrating that IL-4-mediated reduction in GJB2 levels enhanced HIV-1 infection in myeloid cells. Interestingly, GJB2 expression was regulated by IL-4 but not by interferons. The reduction in GJB2 levels was inversely correlated with increased HIV-1 infection levels, suggesting the potential of GJB2 for combating HIV/AIDS.

In respiratory airway epithelial cells, lipopolysaccharide (LPS) treatment induced an enhancement of connexin 26 (Cx26) hemichannel activity shown by dye uptake experiments after siRNA-mediated knock-down of Cx26. This effect was already observed at infection relevant concentrations (≤ 10 ng/mL LPS) and involved tumor necrosis factor alpha (TNF-α)- and Ca 2+ -dependent signaling. High concentrations (1 µg/mL LPS) reduced the transepithelial electrical resistance (TEER) of Calu-3 cells by 35% within an application time of 3 h followed by a recovery. Parallel to barrier alteration, a reduced tight junction organization rate (TiJOR) of claudin-4 (CLDN4) by 75% was observed within an application time of 3 h. After TEER recovery, CLDN4 TiJOR stayed reduced. Low concentrations (10 ng/mL LPS) required three times repeated application for barrier reduction and CLDN4 TiJOR reduction by 30%. The small molecule CVB4-57, newly published as a potential inhibitor of Cx26 hemichannels, mitigated the effects of LPS on the epithelial barrier function. Molecular docking studies revealed a potential interaction between CVB4-57 and Cx26 thereby reducing its hemichannel activity. We conclude that LPS-related enhancement of Cx26 hemichannel activity acts like a “molecular scar” that weakens the lung epithelium, which could be attenuated by agents targeting Cx26 hemichannels.

The gene, which encodes connexin 26 (Cx26), is recognized as the leading cause of non-syndromic hereditary hearing loss. In clinical settings, a total of 131 Cx26 mutations have been identified in association with hearing loss. Certain Cx26 mutants display normal structural and functional properties but fail to translocate to the plasma membrane. Enhancing the membrane localization of these mutants may provide a promising strategy for rescuing hearing loss and hair cell degeneration. This study investigated the membrane localization of Cx26 using cell lines, cultured cochlear explants, and murine models. Key proteins involved in the membrane localization of Cx26 were identified and validated through immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP). Additionally, cell lines and murine models harboring Cx26 mutants were developed to evaluate the effects of Narciclasine on enhancing the membrane localization of these mutants, as well as its potential to rescue hearing loss. The membrane localization of Cx26 was dependent on the integrity of the intracellular transport network consisting of microtubules, actin microfilaments, and the Golgi apparatus. Additionally, SPTBN1 played a significant role in this process. The transmembrane domain 4 (TM4) region exhibited a strong association with the membrane localization of Cx26, and Cx26 mutants located in TM4 region retained in the cytoplasm. Narciclasine promoted cytoskeletal development, thereby enhancing the membrane localization of Cx26 mutants retained in the cytoplasm. This process helped to reconstruct the inner ear gap junction network and rescue hearing loss and hair cell degeneration. These findings present that enhancing the membrane localization of Cx26 mutants can significantly improve auditory function. This strategy offers a potential therapeutic approach for addressing hereditary sensorineural hearing loss associated with mutations.

Inherited hearing impairment is a remarkably heterogeneous monogenic condition, involving hundreds of genes, most of them with very small (< 1%) epidemiological contributions. The exception is GJB2 , the gene encoding connexin-26 and underlying DFNB1, which is the most frequent type of autosomal recessive non-syndromic hearing impairment (ARNSHI) in most populations (up to 40% of ARNSHI cases). DFNB1 is caused by different types of pathogenic variants in GJB2 , but also by large deletions that keep the gene intact but remove an upstream regulatory element that is essential for its expression. Such large deletions, found in most populations, behave as complete loss-of-function variants, usually associated with a profound hearing impairment. By using CRISPR-Cas9 genetic edition, we have generated a murine model ( Dfnb1 em274 ) that reproduces the most frequent of those deletions, del( GJB6 -D13S1830). Dfnb1 em274 homozygous mice are viable, bypassing the embryonic lethality of the Gjb2 knockout, and present a phenotype of profound hearing loss (> 90 dB SPL) that correlates with specific structural abnormalities in the cochlea. We show that Gjb2 expression is nearly abolished and its protein product, Cx26, is nearly absent all throughout the cochlea, unlike previous conditional knockouts in which Gjb2 ablation was not obtained in all cell types. The Dfnb1 em274 model recapitulates the clinical presentation of patients harbouring the del( GJB6 -D13S1830) variant and thus it is a valuable tool to study the pathological mechanisms of DFNB1 and to assay therapies for this most frequent type of human ARNSHI.