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Summary: The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks by gap junction coupling, mainly composed of two gap junction channel proteins, connexin 30 (Cx30) and connexin 43 (Cx43). To circumvent developmental perturbations and to test whether astrocytic gap junction coupling is required for hippocampal neural circuit function and behavior, we generate and study inducible, astrocyte-specific Cx30 and Cx43 double knockouts. Surprisingly, disrupting astrocytic coupling in adult mice results in broad activation of astrocytes and microglia, without obvious signs of pathology. We show that hippocampal CA1 neuron excitability, excitatory synaptic transmission, and long-term potentiation are significantly affected. Moreover, behavioral inspection reveals deficits in sensorimotor performance and a complete lack of spatial learning and memory. Together, our findings establish that astrocytic connexins and an intact astroglial network in the adult brain are vital for neural homeostasis, plasticity, and spatial cognition.

Many of the mutations in GJB2 and GJB6, which encode connexins 26 and 30 (Cx26 and Cx30), impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. In cochlear non-sensory supporting cells, Cx26 and Cx30 form two types of homomeric and heteromeric gap junctions. The biogenesis processes of these channels occurring in situ remain largely unknown. Here we show that Cx30 homomeric and Cx26/Cx30 heteromeric gap junctions exhibit distinct assembly mechanisms in the cochlea. When expressed as homomeric channels, Cx30 preferentially interacts with β-actin in the peripheral non-junctional membrane region, called perinexus, and strongly relies on the actin network for gap junction plaque assembly. In contrast, we found that Cx26/Cx30 heteromeric gap junction plaques are devoid of perinexus and associated actin network, and resist to actin-depolymerizating drug. This supports that Cx26/Cx30 oligomers could be directly delivered from the interior of the cell to the junctional plaque. Altogether, our data provide a novel insight in homomeric and heteromeric gap junction plaque assembly in the cochlea., (Copyright © 2018. Published by Elsevier B.V.)

Mutations in gap junction (GJ) family of proteins, especially in the connexin (Cx) 26, are responsible for causing severe congenital hearing loss in a significant portion of patients (30-50% in various ethnic groups). Substitution of glycine at the position 45 of Cx26 to glutamic acid (p.G45E mutation) causes the Keratitis-ichthyosis-deafness (KID) syndrome. Previous studies have suggested that this point mutation caused a gain-of-function defect. However, the molecular mechanism of KID syndrome remains unclear. Since glycine at this position is conserved in many Cxs expressed in the cochlea, we tested the hypothesis that glycine at position 45 is an important component of the sensor regulating the Ca(2+) gating of GJ hemichannels. Using reconstituted Cx30, 32 and 43 expressed in the HEK 293 cells, we compared the functions of wild type and p.G45E mutant Cxs. We found that G45E in Cx30 resulted in similar deleterious cellular effects as Cx26 did. Cell death occurred within 24h of transfection, which was rescued by increasing extracellular Ca(2+) concentration ([Ca(2+)]o). Dye loading assay showed that Cx30 G45E, similar to Cx26 G45E, had leaky hemichannels at physiological [Ca(2+)]o (1.2 mM). Higher [Ca(2+)]o reduced the dye loading in a dose-dependent manner. Whole cell membrane current recordings also indicated that G45E caused increased hemichannel activities. p.G45E mutations of Cx32 and 43 also resulted in leaky hemichannels compared to their respective wild types in lower [Ca(2+)]o. Our data in this study provided further support for the hypothesis that glycine at position 45 is a conserved Ca(2+) sensor for the gating of GJ hemichannels among multiple Cx subtypes expressed in the cochlea., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Clouston syndrome is a rare autosomal dominant condition characterized by hypotrichosis, nail dystrophy, and occasionally palmoplantar keratoderma. The disease is caused by mutations in GJB6 gene, which encodes a gap junction protein connexin 30 (Cx30)., Objective: To disclose the molecular basis of Clouston syndrome in a Lebanese-German family, and also to determine precise expression of Cx30 in normal skin of humans and mice, as well as transcriptional regulation for the GJB6 expression., Methods: We searched for mutations in the GJB6 gene using DNA of the family members with Clouston syndrome. We performed immunostaining to localize the Cx30 expression in normal human skin and mouse embryos. In addition, we did a series of in vitro studies to investigate if the GJB6 could be a direct transcriptional target gene of p63., Results: We identified a recurrent heterozygous mutation c.31G>C (p.Gly11Arg) in the GJB6 gene in the Lebanese-German family with Clouston syndrome. Immunostaining in normal human skin sections demonstrated predominant expression of Cx30 in hair follicles, nails, and palmoplantar epidermis, which partially overlapped with p63 expression. We also showed co-expression of Cx30 and p63 in developing mouse hair follicles and nail units. In cultured cells, the GJB6 expression was significantly upregulated by ΔNp63α isoform. Further in vitro analyses suggested that ΔNp63α was potentially involved in the GJB6 expression via binding to the sequences in intron 1 of the GJB6 gene., Conclusion: Our data further underscore the crucial roles of Cx30 in morphogenesis and development of skin and its appendages., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Purpose: The present study was initiated to establish the etiological causes of early onset hearing loss (HL) among Estonian children between 2000-2009., Methods: The study group consisted of 233 probands who were first tested with an arrayed primer extension assay, which covers 199 mutations in 7 genes (GJB2, GJB6, GJB3, SLC26A4, SLC26A5 genes, and two mitochondrial genes - 12S rRNA, tRNASer(UCN)). From probands whose etiology of HL remained unknown, DNA analysis of congenital cytomegalovirus (CMV) infection and G-banded karyotype and/or chromosomal microarray analysis (CMA) were performed., Results: In 110 (47%) cases, the etiology of HL was genetic and in 5 (2%) congenital CMV infection was diagnosed. We found mutations with clinical significance in GJB2 (100 children, 43%) and in 2 mitochondrial genes (2 patients, 1%). A single mutation in SLC26A4 gene was detected in 5 probands (2.2%) and was considered diagnostic. In 4 probands a heterozygous IVS2-2A>G change in the SLC26A5 gene was found. We did not find any instances of homozygosity for this splice variant in the probands. CMA identified in 4 probands chromosomal regions with the loss of one allele. In 2 of them we were able to conclude that the found abnormalities are definitely pathogenic (12q13.3-q14.2 and 17q22-23.2 microdeletion), but the pathogenity of 2 other findings (3p26.2 and 1p33 microdeletion) remained unknown., Conclusion: This practical diagnostic algorithm confirmed the etiology of early onset HL for 115 Estonian patients (49%). This algorithm may be generalized to other populations for clinical application.

Gene expression studies often require inducible coexpression of both a gene of interest and a reporter gene. Fusion of fluorescent reporters can, however, modify protein structure and function. We have generated inducible expression systems for two connexin genes: Cx30 and Cx43. It has been reported recently that reporter fusion to connexins can modify their function. Therefore, we compared two methods of independent reporter coexpression and examined colocalization with induced connexin expression. Identical levels of connexin expression were observed for both the bidirectional and bicistronic expression systems. In contrast, however, reporter gene expression by the bidirectional promoter provided brighter average fluorescent pixel intensity than expression of a reporter gene in a bicistronic transcript. Moreover, as a result of this difference in reporter expression, bidirectional expression systems provided equal or better colocalization between the connexins and reporter gene fluorescence. The results of our study indicate that bidirectional reporter expression provides a robust indicator of transfection and gene expression and, therefore, may favor the use of bidirectional over bicistronic reporters in the design of expression systems where the gene of interest, such as a connexin gene, contains translational motifs or long intronic regions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mutations in Gjb2 and Gjb6 genes, coding for connexin26 (Cx26) and Cx30 proteins, respectively, are linked to about half of all cases of human autosomal non-syndromic prelingual deafness. Molecular mechanisms of the hearing impairments, however, are unclear. Most cochlear gap junctions (GJs) are co-assembled from Cx26 and Cx30 and deletion of either one of them causes deafness. Our previous studies have shown that normal hearing is possible in the absence of the Cx30 gene when Cx26 is over-expressed. To further test unique functional requirements for various types of connexins in the hearing, we investigated whether the hearing in the conditional Cx26 (cCx26) null mice could be rescued by genetically over-expressing Cx30. Multiple lines of control and experimental mouse models were used. Auditory brainstem response (ABR) measurements showed normal hearing in targeted gene deletion mice when the deleted Cx26 or Cx30 was transgenically expressed from integrated bacterial artificial chromosome (BAC), demonstrating the effectiveness of the BAC rescue approach. In contrast, severe hearing loss was found in cCx26 null mice in which Cx30 was over-expressed. Morphology observations were consistent with the ABR data. Cochleae of cCx26 null mice with and without the transgenic over-expression of Cx30 both showed the typical immature feature of postnatal cochlear development-the closed tunnel of Corti. Immunolabeling data and Western blot quantification indicated that the Cx26 protein expression preceded that of Cx30 during the early postnatal period in the cochlea. Null expression of Cx26 may therefore uniquely result in a transient period when a total elimination of GJs in functionally-important regions of the developing cochlea is possible. We conclude that Cx26 plays an essential role in the development of the auditory sensory epithelium and its unique developmental functions required for normal hearing is not replaceable by Cx30., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Functional gap junction channels composed of certain connexin proteins are essential for the function of the cochlea. Homozygous deficiency in the Gjb2 (mice) or GJB2 (human) gene coding for connexin26 (Cx26) in the cochlea leads to hearing impairment in mice and humans, respectively. Here we have studied the functional equivalence of Cx26 and connexin32 (Cx32) isoforms in the cochlea. We analyzed a conditional mouse mutant in which the Gjb2 coding DNA was exchanged by LacZ DNA coding for the reporter protein beta-galactosidase. This allowed us to follow the unrestricted and cell type specific expression of Gjb2 promoter activity. After inner ear specific, Otogelin-Cre recombinase mediated deletion of the loxP-site-flanked LacZ coding DNA, transcription of the Gjb1 gene, coding for Cx32 was activated by the Gjb2 promoter. Interbreeding of these mice with conditional Gjb2 null mice resulted in animals in which Cx32 instead of Cx26 protein is expressed in the non-sensory epithelial network of the cochlea. When we analyzed the auditory function in these mice, we found that the expression of Cx32 protein is sufficient to support hearing in the absence of Cx26. Thus Cx32 can functionally replace Cx26 in the mouse cochlea resulting in almost normal hearing., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Background: Suicide is an important public health problem that results from the interaction of both psychosocial and biological factors. Although it is known that particular neurobiological processes underlie suicidal ideation and behavior, there still remains limited knowledge about the specific factors involved., Methods: To explore the neurobiology of suicide we generated microarray data from dorsal lateral prefrontal cortex (DLPFC) in each of 28 male French-Canadian subjects (20 suicide completers). These results were followed up in a larger French-Canadian sample (n = 47, 38 suicide completers) and in microarray data available from the Stanley Foundation (n = 100, 36 suicide completers). To investigate the molecular mechanisms of this finding, we performed RNA interference and electrophoretic mobility shift assays. Animal behavioral experiments were done to control for drug and alcohol effects., Results: We found reduced expression of Cx30 and Cx43 in DLPFC of suicide completers. We identified a previously unknown function for Sox9 as a transcription factor affecting expression of Cx30 in brain., Conclusions: These results suggest that alterations of astrocyte connexins might be involved in the suicide process and provide further evidence implicating astrocytes in psychopathology., (Copyright © 2011 Society of Biological Psychiatry. All rights reserved.)

Acute hypoxia at postnatal day (P) 10 is an accepted model of human neonatal hypoxia which results, among other consequences, in increased hippocampal excitability. Hypoxic-ischemic injury, which mimics stroke, has been shown to result in changes in connexins (Cxs), however, changes in Cxs have not been studied in the P10 hypoxia model. The aim of this study was to investigate changes in the hippocampal expression of three different connexins at consecutive developmental stages after acute hypoxia at P10 (10min and 30min after reoxygenation, P11, P14, P17, P29, and P45) as compared to sham manipulated pups. After acute hypoxia at P10, Cx30 protein levels were increased at 30min after reoxygenation, at P11 and at P14, and then returned to control levels. Cx36 protein levels transiently decreased at P11 after acute hypoxia then returned to control levels. Cx43 protein levels did not change at any of the time points. Although changes in mRNA expression were observed during development for Cx30 only, acute hypoxia did not result in changes in mRNA expression of all these Cxs when compared to age matched controls suggesting that acute hypoxia induced posttranslational changes in protein expression., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Purpose: Usher syndrome is a major cause of genetic deafness and blindness. The hearing loss is usually congenital and the retinitis pigmentosa is progressive and first noticed in early childhood to the middle teenage years. Its frequency may be underestimated. Newly developed molecular technologies can detect the underlying gene mutation of this disorder early in life providing estimation of its prevalence in at risk pediatric populations and laying a foundation for its incorporation as an adjunct to newborn hearing screening programs., Methods: A total of 133 children from two deaf and hard of hearing pediatric populations were genotyped first for GJB2/6 and, if negative, then for Usher syndrome. Children were scored as positive if the test revealed > or =1 pathogenic mutations in any Usher gene., Results: Fifteen children carried pathogenic mutations in one of the Usher genes; the number of deaf and hard of hearing children carrying Usher syndrome mutations was 15/133 (11.3%). The population prevalence was estimated to be 1/6000., Conclusion: Usher syndrome is more prevalent than has been reported before the genome project era. Early diagnosis of Usher syndrome has important positive implications for childhood safety, educational planning, genetic counseling, and treatment. The results demonstrate that DNA testing for Usher syndrome is feasible and may be a useful addition to newborn hearing screening programs.

Connexin 26 and connexin 30 are the major connexins expressed in the cochlea, where they are co-localized and form heteromeric gap junctions. Mutations in the GJB2 gene, which encodes connexin 26, are the most common cause of prelingual non-syndromic sensorineural hearing loss. The large del(GJB6-D13S1830) mutation which involves GJB6 (connexin 30), causes hearing loss in homozygous individuals, or when compound heterozygous with a GJB2 mutation. Until now, it remained unresolved whether this phenomenon results from digenic inheritance or because of lack of GJB2 mRNA expression. After RNA extraction from buccal epithelium, a tissue known to express connexin 26 as well as connexin 30, allele-specific expression of GJB2 was investigated by reverse-transcriptase PCR and restriction digestions in three unrelated individuals compound heterozygous for a GJB2 mutation and del(GJB6-D13S1830). Each proband carried a different sequence change in GJB2. The mutated GJB2 allele in trans with del(GJB6-D13S1830) was expressed in all three individuals whereas the GJB2 allele located in cis with the deletion was not expressed at all. Thus, mutations in these two genes do not cause hearing loss through a digenic mechanism of inheritance alone, as was postulated previously, but instead GJB2 expression is abolished through an effect in cis with the deletion. Our study provides unequivocal support for the hypothesis that del(GJB6-D13S1830) eliminates a putative cis-regulatory element located within the deleted region.

Mutations at the DFNB1 locus which encode connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively, are main cause for sporadic and familial non-syndromic hearing impairment (NSHI) in many populations. 342-kb deletion [del (GJB6-D13S1830)] of Cx30 gene is second most common connexin mutation. Specific mitochondrial DNA (mtDNA) mutations have been found to be associated with NSHI. In this study, we screened 210 NSHI patients for GJB2 mutations, DeltaGJB6-D13S1830 deletion and three point mutations in mtDNA (A1555G, A3243G, A7445G) using PCR, DHPLC and sequencing in North Indian cohort. 35delG was found to be the most common mutation (10.9%), followed by W24X (3.8%) and W77X (1.9%) mutations. We did not observe GJB6-D13S1830 deletion and three mitochondrial point mutations in our cohort. Most of patients (50/58) carried monoallelic variations. Our results reveal different spectrum of GJB2 mutations specific to North Indian cohort, with 35delG being most prevalent. These results suggest that different types of GJB2 mutations affect autosomal recessive NSHI according to ethnic background.

Biallelic mutations in the GJB2, GJB3, GJB6 and CLDN14 genes have been implicated in autosomal recessive non-syndromic hearing impairment (ARNSHI). Moreover, a large number of GJB2 heterozygous patients was reported. The phenotype was in partly justified by the occurrence of two deletions including GJB6. We analysed GJB2, GJB6, GJB3 and CLDN14 in 102 Tunisian patients with ARNSHI. The deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) were also screened. The c.35delG in GJB2 was the most frequent mutation (21.57%). It was detected at heterozygous state in 2 patients. The del(GJB6-D13S1830) was identified in one case at heterozygous state. No other mutation in studied gap junction genes was detected in heterozygous patients. Several polymorphisms were identified in GJB3, GJB6 and CLDN14. Our study confirms the importance of GJB2 screening in ARNSHI and suggests that in consanguineous populations, a single DFNB1 mutant allele in individuals with HI is likely due to a coincidental carrier state.

A systematic approach, involving haplotyping and genotyping, to the molecular diagnosis of non-syndromic deafness within 50 families and 9 sporadic cases from Algeria is described. Mutations at the DFNB1 locus (encompassing the GJB2 and GJB6 genes) are responsible for more than half of autosomal recessive prelingual non-syndromic deafness in various populations. A c.35delG mutation can account for up to 85% of GJB2 mutations and two large deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) have also been reported in several population groups. In view of the genetic heterogeneity a strategy was developed which involved direct analysis of DFNB1. In negative familial cases, haplotype analysis was carried out, where possible, to exclude DFNB1 mutations. Following this, haplotype analysis of five Usher syndrome loci, sometimes involved in autosomal non-syndromic hearing loss, was carried out to identify cases in which Usher gene sequencing was indicated. When homozygosity was observed at a locus in a consanguineous family, the corresponding gene was exhaustively sequenced. Pathogenic DFNB1 genotypes were identified in 40% of the cases. Of the 21 cases identified with 2 pathogenic mutations, c.35delG represented 76% of the mutated alleles. The additional mutations were one nonsense, two missense and one splicing mutation. Four additional patients were identified with a single DFNB1 mutation. None carried the large deletions. Three families with non-syndromic deafness carried novel unclassified variants (UVs) in MYO7A (1 family) and CDH23 (2 families) of unknown pathogenic effect. Additionally, molecular diagnosis was carried out on two Usher type I families and pathogenic mutations in MYO7A and PCDH15 were found.

Purpose: To identify factors that are associated with why parents of deaf children who have had GJB2/GJB6 testing as part of a genetics research study do or do not take their children for genetics evaluation., Methods: Self-administered questionnaire was completed by parents of a deaf child participating in a GJB2/GJB6 testing study., Results: A total of 30 parents (representing 24 children) completed the questionnaire; 11 of 24 children (46%) underwent a genetics evaluation. Compared with parents who did not take their child for a genetics evaluation, those who did were more likely to (1) have supportive pediatricians, (2) feel it was important or would be helpful to their child, (3) recall the recommendation for evaluation, (4) have family members who wanted the child to have an evaluation, and (5) be Hispanic or Asian. Genetic test results, knowledge of genetics evaluation, psychosocial factors, language concerns, or structural factors were not substantively associated with attending a genetics evaluation., Conclusion: Parental perceptions, family environment, and pediatricians play a role in decisions regarding genetics evaluation. Because genetic testing for deafness likely will occur outside of traditional genetics clinics and without comprehensive genetics evaluation, efforts to increase pediatricians' awareness of the usefulness of genetics evaluation may be essential to ensure appropriate care for deaf and hard-of-hearing children as recommended by the American College of Medical Genetics.

Purpose: Previous studies of connexin-related hearing loss have typically reported on mixed age groups or adults. To further address epidemiology and natural history of connexin-related hearing loss, we conducted a longitudinal study in an ethnically diverse cohort of infants and toddlers under 3 years of age. Our study compares infants with and without connexin-related hearing loss to examine differences in the prevalence of connexin and non-connexin-related hearing loss by ethnic origin, detection by newborn hearing screening, phenotype, neonatal risk factors, and family history. This is the first study to differentiate infants with and without connexin-related hearing loss., Methods: We enrolled 95 infants with hearing loss from whom both exons of Cx26 were sequenced and the Cx30 deletion was assayed. Demographic, family history, newborn hearing screening data, perinatal, and audiologic records were analyzed., Results: Genetic testing identified biallelic Cx26/30 hearing loss-associated variants in 24.7% of infants with a significantly lower prevalence in Hispanic infants (9.1%). Eighty-two infants underwent newborn hearing screening; 12 infants passed, 3 had connexin-related hearing loss. No differences in newborn hearing screening pass rate, neonatal complications, or hearing loss severity were detected between infants with and without connexin-related hearing loss. Family history correlates with connexin-related hearing loss., Conclusions: Connexin-related hearing loss occurs in one quarter of infants in an ethnically diverse hearing loss population but with a lower prevalence in Hispanic infants. Not all infants with connexin-related hearing loss fail newborn hearing screening. Family history correlates significantly with connexin-related hearing loss. Genetic testing should not be deferred because of newborn complications. These results will have an impact on genetic testing for infant hearing loss.

Three-dimensional (3D) organotypic models are increasingly used to study the aspects of epidermal organisation and cutaneous wound-healing events. However, these are largely dependent on laborious histological analysis and immunohistochemical approaches. Despite the large resource of transgenic and knockout mice harboring mutations relevant to skin disorders, few organotypic mouse skin models are available. We have developed a versatile in vitro 3D organotypic mouse skin equivalent that reflects epidermal organisation in vivo. The system is optically transparent and ideally suited to real-time analysis using a variety of integrated in situ imaging techniques. As a paradigm for coordination of cellular events, the epidermal gap junction network was investigated and the model displayed predominant connexin 43 (Cx43) expression in basal proliferating cells and Cx26 and Cx30 expression in differentiated keratinocytes. We show that attenuation of Cx43-mediated communication by a Cx mimetic peptide enhanced wound closure rates in keratinocyte monocultures and in the living skin equivalent system, emphasising the utility of the model to systematically unravel the molecular mechanisms underlying epidermal morphogenesis, assess promising therapeutic strategies, and reduce animal experimentation. Furthermore, we visualise epidermal regeneration following injury in real time, thereby facilitating avenues to explore distinctive modes of wound re-epithelialisation in a non-invasive manner.

Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening., Methods: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort., Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants., Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.

Gap junctions are documented in the human airway epithelium but the functional expression and molecular identity of their protein constituents (connexins, Cx) in the polarized epithelium is not known. To address this question, we documented the expression of a family of epithelial Cx (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, and Cx43) in primary human airway epithelial cells (AEC) grown on porous supports. Under submerged conditions, AEC formed a monolayer of airway cells whereas the air-liquid interface induced within 30-60 days AEC differentiation into a polarized epithelium for up to 6-9 months. Maturation of AEC was associated with the down-regulation of Cx26 and Cx43. The well-differentiated airway epithelium exhibited gap junctional communication between ciliated and between ciliated and basal cells. Interestingly, Cx30 was mostly present between ciliated cells whereas Cx31 was found between basal cells. These results are supportive of the establishment of signal-selective gap junctions with maturation of AEC, likely contributing to support airway epithelium function. These results lay the ground for studying the role of Cx-mediated cell-cell communication during repair following AEC injury and exploring Cx-targeted interventions to modulate the healing process.

Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays > 60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.

Connexins are integral membrane proteins forming aqueous gap junction channels that allow the diffusional exchange of ions and small metabolites between cells, thus coordinating metabolic activities in multicellular tissues. Dominant mutations in the Cx26 gene GJB2 have been shown to cause keratitis-ichthyosis-deafness (KID) syndrome, palmoplantar keratoderma associated with hearing loss, and Vohwinkel syndrome. Missense mutations in the closely related Cx30 gene GJB6 underlie Clouston syndrome (autosomal dominant hidrotic ectodermal dysplasia). We report a 6-y-old boy with phenotypic characteristics of KID syndrome as well as atrichia. In contrast to other KID syndrome patients, molecular analysis of the connexin gene GJB2 did not disclose a pathogenic mutation, although the patient was homozygous for a common polymorphism (V27I) in the coding sequence of Cx26. Nevertheless, screening of GJB6 revealed a heterozygous missense mutation (V37E) predicted to alter sequence and charge of the first transmembrane helix of Cx30, which was previously implicated in Clouston syndrome (Smith et al, 2002). The presence of a pathogenic Cx30 mutation and the lack of a pathologic molecular change in Cx26 in this patient, whose clinical features predominantly resemble KID syndrome, suggest genetic heterogeneity of KID syndrome and underscore that mutations in Cx30, similar to those in Cx26 or Cx31, can cause different phenotypes. Based on our results, connexin gene mutations should be considered in patients presenting with congenital sensorineural hearing loss and disorders of cornification, and screening of several connexin genes with known cutaneous phenotype, such as those for Cx26, Cx30, Cx30.3, and Cx31, may be required.

Gap junctions (GJ) are known to be involved in spontaneous wound healing in rodent skin. We analyzed the staining patterns of the GJ proteins Cx26, Cx30, and Cx43 in human cutaneous wound healing and compared ex vivo spontaneous wound healing to non-healing wounds (chronic leg ulcers) and to ex vivo accelerated wound healing after transplantation of cultured keratinocytes. We demonstrate a loss of Cx43 staining at the wound margins during initial wound healing and after transplantation of keratinocytes. In contrast, Cx43 remains present at the margins of most non-healing wounds. We show a subsequent induction of Cx26 and Cx30 near the wound margins in spontaneous wound healing and-even earlier-after the transplantation of keratinocytes. The cells at the wound margins remain negative until the commencement of epidermal regeneration. Cx26/30 are present at the wound margins of most non-healing wounds. Cx stainings are absent in the transplanted keratinocytes during early wound healing, but there is a subsequent induction. Our results suggest that the downregulation of Cx43 is an important event in human wound healing. We discuss the assumption that direct cell-cell communication via GJ contribute to the acceleration of wound healing after the transplantation of keratinocytes.

We studied three families suffering from nail abnormalities who had previously been diagnosed as pachyonychia congenita. No keratin gene mutations were detected. Sequencing of connexin 30 (GJB6 gene) in these patients identified heterozygous missense mutations G11R and A88V that are known to be associated with Clouston syndrome. This unexpected finding expands the Clouston syndrome phenotype and suggests that some patients diagnosed with pachyonychia may in fact be suffering from Clouston syndrome.

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.

Purpose: Profound hearing loss occurs with a frequency of 1 in 1000 live births, half of which is genetic in etiology. The past decade has witnessed rapid advances in determining the pathogenesis of both syndromic and nonsyndromic deafness. The most significant clinical finding to date has been the discovery that mutations of GJB2 at the DFNB1 locus are the major cause of profound prelingual deafness in many countries. 1 More recently, GJB2 mutations have been shown to cause deafness when present with a deletion of the GJB6 gene. We report on the prevalence of GJB2 and GJB6 mutations in a large North American Repository of DNA from deaf probands and document the profound effects of familial ethnicity and parental mating types on the frequency of these mutations in the population., Methods: Deaf probands were ascertained through the Annual Survey of Deaf and Hard of Hearing Children and Youth, conducted at the Research Institute of Gallaudet University. Educational, etiologic, and audiologic information was collected after obtaining informed consent. DNA studies were performed for the GJB2 and GJB6 loci by sequencing and PCR methods., Results: GJB2 mutations accounted for 22.2% of deafness in the overall sample but differed significantly among Asians, African-Americans and Hispanics and for probands from deaf by deaf and deaf by hearing matings, as well as probands from simplex and multiplex sibships of hearing parents. In our sample, the overall incidence of GJB2/GJB6 deafness was 2.57%., Conclusion: GJB2 mutations account for a large proportion of deafness in the US, with certain mutations having a high ethnic predilection. Heterozygotes at the GJB2 locus should be screened for the GJB6 deletion as a cause of deafness. Molecular testing for GJB2 and GJB6 should be offered to all patients with nonsyndromic hearing loss.

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26-EYFP fusion products was comparable to that of hCx26-pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30-EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26-EYFP and hCx30-EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26-pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg(j)) displayed significantly smaller values (28.8+/-1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5+/-3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (G(j)) on transjunctional voltage (V(j)). The half-activation voltage for G(j) was close to +/-95 and +/-58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tg(j)= 45.2 +/- 3.5 nS and V(0)= +/- 34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies.

Background: hidrotic ectodermal dysplasia (HED) or Clouston syndrome is a rare autosomal dominant disorder affecting the skin and its derivatives. It is characterized by the triad of nail dystrophy, alopecia, and palmoplantar hyperkeratosis. To date, all mutations have been involving in three codons: G11R, A88V and V37E in the connexin 30 (Cx30) gene have been shown to cause this disorder., Objective: in order to analyze the mutations of the Cx30 gene in Chinese Han patients with HED., Methods: we collected a large Chinese HED family consisting of a total of 81 individuals including 28 HED patients (14 males and 14 females). The whole coding region of Cx30 was amplified by polymerase chain reaction and products analyzed by direct sequencing, then further confirmed at the mRNA level by RT-PCR., Results: we detected a transition, 31(G-->A), leading to a missense mutation (G11R) in genomic DNAs of 18 patients, and the point mutation was not found in 16 normal individuals in this HED family and in 188 unrelated, population-match control individuals. The transcription of mutated allele was confirmed by RT-PCR of Cx30 mRNA., Conclusion: our data suggests that a G11R missense mutation in the Cx30 gene can cause HED in Chinese Han population and emphasizes the importance of screening for this as well as other Cx30 gene mutations in the HED.

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.

Clouston syndrome (hidrotic ectodermal dysplasia) is an autosomal dominant ectodermal dysplasia characterized by alopecia, palmoplantar hyperkeratosis, and nail dystrophy. Recently, mutations in the GJB6 gene encoding the gap junction protein connexin 30 have been shown to cause this disorder. To date, all mutations have involved two codons: G11R and A88V. Here, we report a novel mutation V37E within the first transmembrane domain of connexin 30 in a spontaneous case of Clouston syndrome. The mutation was detected in genomic DNA, confirmed in reverse transcription polymerase chain reaction products, and was excluded from 100 ethnically matched control individuals by restriction enzyme analysis.