Cx26 Mutations Research Articles

Keratitis-Ichthyosis-Deafness Syndrome-Associated Cx26 Mutants Produce Nonfunctional Gap Junctions but Hyperactive Hemichannels When Co-Expressed With Wild Type Cx43

Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin.

Functional hemichannels formed by human connexin 26 expressed in bacteria.

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure–function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.

Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures.

Gja1(Jrt)/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1(Jrt)/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1(Jrt)/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1(Jrt)/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1(Jrt)/+ osteoblasts and increased Pparg2 expression in bone marrow-derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1(Jrt)/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1(Jrt)/+ mouse model.

Connexins: Intercellular Signal Transmitters in Lymphohematopoietic Tissues.

Life-long hematopoietic demands are met by a pool of hematopoietic stem cells (HSC) with self-renewal and multipotential differentiation ability. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment control HSC activity. Cell-to-cell communication through connexin (Cx) containing gap junctions (GJs) allows pluricellular coordination and synchronization through transfer of small molecules with messenger activity. Hematopoietic and surrounding nonhematopoietic cells communicate each other through GJs, which regulate fetal and postnatal HSC content and function in hematopoietic tissues. Traffic of HSC between peripheral blood and BM is also dependent on Cx proteins. Cx mutations are associated with human disease and hematopoietic dysfunction and Cx signaling may represent a target for therapeutic intervention. In this review, we illustrate and highlight the importance of Cxs in the regulation of hematopoietic homeostasis under normal and pathological conditions.

AMP-Activated Protein Kinase α1 Regulates Cardiac Gap Junction Protein Connexin 43 and Electrical Remodeling Following Pressure Overload

Background/Aims: Adenosine 5'-monophosphate (AMP)-activated protein kinase (Ampk) modulates a wide array of cellular functions and regulates various ion channels and transporters. In failing human hearts an increased Ampkα1 activity was observed. The present study aimed to uncover the impact of Ampkα1 on cardiac electrical remodeling. Methods: Gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and corresponding wild-type mice were exposed to pressure overload by “transverse aortic constriction” (TAC). In vivo electrophysiology was performed with a single catheter technique, myocardial conduction velocities and conduction characteristics investigated in isolated hearts, transcript levels quantified by RT-PCR and protein abundance determined by Western blotting. Moreover, connexin 43 (Cx43) was expressed in Xenopus oocytes with or without coexpression of wild-type or mutant AMPK and Cx43 protein abundance quantified utilizing confocal microscopy. Results: TAC treatment increased Ampkα1 protein expression in cardiac tissue from wild-type mice. TAC further increased left ventricular conduction inhomogeneity and triggered conduction blocks, effects blunted in the Ampkα1^-/- mice. TAC treatment decreased Cx43 protein abundance in cardiac tissue, an effect significantly blunted in the Ampkα1^-/- mice. TAC treatment did not modify Cx43 mRNA levels but increased ubiquitination of Cx43 protein, an effect mitigated by Ampkα1 deficiency. As shown in Xenopus oocytes, Cx43 cell membrane protein abundance was significantly downregulated by wild-type AMPK^WT and constitutively active AMPK^γR70Q, but not by catalytically inactive AMPK^αK45R. Conclusion: Ampkα1 stimulates ubiquitination of the gap junction protein Cx43, thereby contributing to gap junction remodeling following pressure overload.

Understanding of the molecular evolution of deafness-associated pathogenic mutations of connexin 26.

Connexins (Cxs) were first identified as subunit proteins of the intercellular membrane channels that cluster in the cell communication structures known as gap junctions. Mutations in the gap junction β2 (GJB2) gene encoding connexin 26 (Cx26) have been linked to sporadic and hereditary hearing loss. In some cases, the mechanisms through which these mutations lead to hearing loss have been partly elucidated using cell culture systems and animal models. The goal of this study was to re-assess the pathogenic roles of the GJB2 mutations by combining comparative evolutionary studies. We used Bayesian phylogenetic analyses to determine the relationships among 35 orthologs and to calculate the ancestral sequences of these orthologs. By aligning sequences from the 35 orthologs and their ancestors and categorizing amino acid sites by degree of conservation, we used comparative evolutionary methods to determine potential functionally important amino acid sites in Cx26 and to identify missense changes that are likely to affect function. We identified six conserved regions in Cx26, five of which are located in the Connexin_CCC, and another is in the connexin super family domain. Finally, we identified 51 missense changes that are likely to disrupt function, and the probability of these changes occurring at hydrophilic amino acid residues was twice that of occurring at hydrophobic residues in the trans-membrane regions of Cx26. Our findings, which were obtained by combining comparative evolutionary methods to predict Cx26 mutant function, are consistent with the pathogenic characteristics of Cx26 mutants. This study provides a new pathway for studying the role of aberrant Cx26 in hereditary hearing loss.

Connexin 43 channels are essential for normal bone structure and osteocyte viability.

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks the gap junction channel, but not the hemichannel function, and the Δ130-136 mutant inhibits activity of both types of channels. Δ130-136 mice showed a significant increase in bone mineral density compared to wild-type (WT) and R76W mice. Micro-computed tomography (µCT) analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130-136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130-136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130-136 mice that likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption, and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.

Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca(2+) signaling in HeLa cells.

Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects.

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.

Reduced expression of Connexin26 and its DNA promoter hypermethylation in the inner ear of mimetic aging rats induced by d-galactose

Connexin26 (Cx26), one of the major protein subunits forming gap junctions (GJs), is important in maintaining homeostasis in the inner ear and normal hearing. Cx26 mutation is one of the most common causes for inherited nonsyndromic deafness, but the relationship between Cx26 and presbycusis is unknown. Our study aimed at exploring the expression and the aberrant methylation of the promoter region of Cx26 gene in the cochlea of inner ear mimetic aging rats. We applied a mimetic aging of inner ear rat model with mtDNA common deletion by d-gal injection for 8weeks. Real-time RT-PCR and Western blot of rat inner ear tissue indicated that the Cx26 expression decreased in the d-gal group. Further bisulfite sequencing analysis revealed that the methylation status of the promoter region of Cx26 gene in the d-gal group was higher than that in control group. These results indicated that the decrease of Cx26 expression might contribute to the development of presbycusis and the hypermethylation of promoter region of GJB2 might be associated with the Cx26 downregulation.

Mammary gland specific knockdown of the physiological surge in Cx26 during lactation retains normal mammary gland development and function.

Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function.

R75Q de novo dominant mutation of GJB2 in a Chinese family with hearing loss and palmoplantar keratoderma

ObjectivesMutations in the GJB2 gene encoding connexin 26 (Cx26) are major causes of hereditary deafness. This study aimed to characterize the mutation profiles of the GJB2 gene in a Chinese family with sensorineural hearing loss. MethodsA Chinese family that included three individuals with sensorineural hearing loss and palmoplantar keratoderma underwent complete physical examinations, audiological examinations including pure tone audiometry and auditory brainstem response, skin pathological examination, and temporal CT scans. The entire coding region of GJB2, GJB3, GJB6, and the coding exons (exon7+8 and 19) of SLC26A4, mitochondrial 12SrRNA, and tRNA Ser (UCN) were sequenced. Structural analysis was performed to detect the effects of mutation on the tertiary structure of Cx26. ResultsA dominant GJB2 mutation, c.224G>A (p.Arg75Gln, p.R75Q), was detected in the family. No other mutation was identified in GJB2, GJB3, GJB6, or the coding exons (exon7+8 and 19) of SLC26A4, mitochondrial 12SrRNA, and tRNA Ser (UCN). Structural analysis revealed that the p.R75Q mutation likely affects the structural stability and permeation properties of the Cx26 gap junction channel. ConclusionOur findings provide further evidence of a correlation between the p.R75Q mutation in Cx26 and a syndromic hearing impairment with palmoplantar keratoderma.

CamKII inhibitors reduce mitotic instability, connexon anomalies and progression of the in vivo behavioral phenotype in transgenic animals expressing a mutated Gjb1 gene.

Mutation in the Gjb1 gene, coding for a connexin (Cx32), is associated with an inherited peripheral neuropathic disorder (X-linked Charcot-Marie-Tooth, CMTX). Our previous work reported that transgenic animals expressing a human Gjb1 transgene present polyploidy and abnormal over-duplication of the centrosome, suggesting a role for Gjb1 in mitotic stability. In this article, we propose mechanisms by which mutations in Gjb1 induce mitotic instability and discuss its potential relation with the CMTX phenotype. We showed that transgenic cells exhibit CamKII over-stimulation, a phenomenon that has been linked to mitotic instability (polyploidy, nuclear volume and centrosome over-duplication), that is reversed by CamKII inhibitors. We also demonstrate that connexon activity is partially restored in transgenic cells with CamKII inhibitors. Our model supports the role for Pim1, a kinase that has been associated with genomic instability in cancers, in genomic instability in Cx32 mutations. Regarding in vivo phenotype, we showed that degradation on the rotarod test in our transgenic mice is significantly lowered by treatment with a CamKII inhibitor (KN93). This effect was seen in two lines with different point mutations in GJB1, and stopping the treatment led to degradation of the phenotype.

The connexin 30.3 of zebrafish homologue of human connexin 26 may play similar role in the inner ear

The intercellular gap junction channels formed by connexins (CXs) are important for recycling potassium ions in the inner ear. CXs are encoded by a family of the CX gene, such as GJB2, and the mechanism leading to mutant connexin-associated diseases, including hearing loss, remains to be elucidated. In this study, using bioinformatics, we found that two zebrafish cx genes, cx27.5 and cx30.3, are likely homologous to human and mouse GJB2. During embryogenesis, zebrafish cx27.5 was rarely expressed at 1.5–3 h post-fertilization (hpf), but a relatively high level of cx27.5 expression was detected from 6 to 96 hpf. However, zebrafish cx30.3 transcripts were hardly detected until 9 hpf. The temporal experiment was conducted in whole larvae. Both cx27.5 and cx30.3 transcripts were revealed significantly in the inner ear by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). In the HeLa cell model, we found that zebrafish Cx27.5 was distributed intracellularly in the cytoplasm, whereas Cx30.3 was localized in the plasma membrane of HeLa cells stably expressing Cx proteins. The expression pattern of zebrafish Cx30.3 in HeLa cells was more similar to that of cells expressing human CX26 than Cx27.5. In addition, we found that Cx30.3 was localized in the cell membrane of hair cells within the inner ear by immunohistochemistry (IHC), suggesting that zebrafish cx30.3 might play an essential role in the development of the inner ear, in the same manner as human GJB2. We then performed morpholino knockdown studies in zebrafish embryos to elucidate the physiological functions of Cx30.3. The zebrafish cx30.3 morphants exhibited wild-type-like and heart edema phenotypes with smaller inner ears at 72 hpf. Based on these results, we suggest that the zebrafish Cx30.3 and mammalian CX26 may play alike roles in the inner ear. Thus, zebrafish can potentially serve as a model for studying hearing loss disorders that result from human CX26 mutations.

A 14-3-3 mode-1 binding motif initiates gap junction internalization during acute cardiac ischemia.

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14-3-3 proteins can regulate protein trafficking, and a 14-3-3 mode-1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C-terminus. We hypothesized that Cx43(Ser373) phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14-3-3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14-3-3τ in cell lines increases gap junction plaque size at cell-cell borders. Cx43(S373A) mutation prevents Cx43/14-3-3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff-perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no-flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen-activated protein kinase has previously been implicated in Cx43 internalization. The Cx43(S373A) mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43(Ser373) phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.

Mutations of Cx43 that affect B cell spreading in response to BCR signaling

ABSTRACTThe gap junction (GJ) protein connexin 43 (Cx43) is both necessary and sufficient for B cell receptor (BCR)-mediated cell spreading. To address how Cx43 mediates this effect, we blocked its function genetically, by expressing mutants of Cx43, and pharmacologically, by using chemical inhibitors. While various point mutations of Cx43 inhibited B cell spreading, treatment with channel blocking drugs did not, suggesting that this response was independent of channel function. The critical region of Cx43 appears to be the cytoplasmic carboxyl-terminal (CT) domain, which has previously been shown to be important for B cell spreading. Consistent with this, mutations of either tyrosine 247 or 265 found in the CT were sufficient to inhibit spreading. Thus Cx43 may influence B cell spreading by mechanisms requiring protein binding to, or modification of, these sites in the CT tail.

Molecular dynamics simulations highlight structural and functional alterations in deafness–related M34T mutation of connexin 26

Mutations of the GJB2 gene encoding the connexin 26 (Cx26) gap junction protein, which is widely expressed in the inner ear, are the primary cause of hereditary non-syndromic hearing loss in several populations. The deafness–associated single amino acid substitution of methionine 34 (M34) in the first transmembrane helix (TM1) with a threonine (T) ensues in the production of mutant Cx26M34T channels that are correctly synthesized and assembled in the plasma membrane. However, mutant channels overexpressed in HeLa cells retain only 11% of the wild type unitary conductance. Here we extend and rationalize those findings by comparing wild type Cx26 (Cx26WT) and Cx26M34T mutant channels in silico, using molecular dynamics simulations. Our results indicate that the quaternary structure of the Cx26M34T hemichannel is altered at the level of the pore funnel due to the disruption of the hydrophobic interaction between M34 and tryptophan 3 (W3) in the N–terminal helix (NTH). Our simulations also show that external force stimuli applied to the NTHs can detach them from the inner wall of the pore more readily in the mutant than in the wild type hemichannel. These structural alterations significantly increase the free energy barrier encountered by permeating ions, correspondingly decreasing the unitary conductance of the Cx26M34T hemichannel. Our results accord with the proposal that the mutant resides most of the time in a low conductance state. However, the small displacement of the NTHs in our Cx26M34T hemichannel model is not compatible with the formation of a pore plug as in the related Cx26M34A mutant.

Down regulated connexin26 at different postnatal stage displayed different types of cellular degeneration and formation of organ of Corti

Connexin26 (Cx26) mutation is the most common cause for non-syndromic hereditary deafness. Different congenital Cx26 null mouse models revealed a profound hearing loss pattern and developmental defect in the cochlea. Our study aimed at establishing a Cx26 knocking down mouse model at different postnatal time points and to investigate the time course and pattern of the hearing loss and cell degeneration in these models. Morphologic changes were observed for 5months to detect long-term diversities among these models. Depending on the time point when Cx26 expression was reduced, mild to profound hearing loss patterns were found in different groups. Malformed organ of Corti with distinct cell loss in middle turn was observed only in early Cx26 reduction group while mice in late Cx26 reduction group developed normal organ of Corti and only suffered a few hair loss in the basal turn. These results indicated that Cx26 may play essential roles in the postnatal maturation of the cochlea, and its role in normal hearing at more mature stage may be replaceable.

Myogenic bladder defects in mouse models of human oculodentodigital dysplasia

To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (oculodentodigital dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43(G60S) and Cx43(I130T)) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43(G60S) mutant mice showed no difference in voided urine volume or frequency, the Cx43(I130T) mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43(I130T) mice are likely to be complemented by neurogenic changes.

Functional analysis and regulation of purified connexin hemichannels

Gap-junction channels (GJCs) are aqueous channels that communicate adjacent cells. They are formed by head-to-head association of two hemichannels (HCs), one from each of the adjacent cells. Functional HCs are connexin hexamers composed of one or more connexin isoforms. Deafness is the most frequent sensineural disorder, and mutations of Cx26 are the most common cause of genetic deafness. Cx43 is the most ubiquitous connexin, expressed in many organs, tissues, and cell types, including heart, brain, and kidney. Alterations in its expression and function play important roles in the pathophysiology of very frequent medical problems such as those related to cardiac and brain ischemia. There is extensive information on the relationship between phosphorylation and Cx43 targeting, location, and function from experiments in cells and organs in normal and pathological conditions. However, the molecular mechanisms of Cx43 regulation by phosphorylation are hard to tackle in complex systems. Here, we present the use of purified HCs as a model for functional and structural studies. Cx26 and Cx43 are the only isoforms that have been purified, reconstituted, and subjected to functional and structural analysis. Purified Cx26 and Cx43 HCs have properties compatible with those demonstrated in cells, and present methodologies for the functional analysis of purified HCs reconstituted in liposomes. We show that phosphorylation of serine 368 by PKC produces a partial closure of the Cx43 HCs, changing solute selectivity. We also present evidence that the effect of phosphorylation is highly cooperative, requiring modification of several connexin subunits, and that phosphorylation of serine 368 elicits conformational changes in the purified HCs. The use of purified HCs is starting to provide critical data to understand the regulation of HCs at the molecular level.