Gap Junction Connexin Research Articles

Structural determinants underlying permeant discrimination of the Cx43 hemichannel

Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 µg/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

The identification and characterization of the p.G91 deletion in CRYBA1 in a Chinese family with congenital cataracts

BackgroundMutations in more than 52 genes have been identified in isolated congenital cataracts, the majority of which are located in crystalline and connexin (gap junction) genes. An in-frame one amino acid deletion in the beta-crystalline gene CRYBA1 has been reported in several different Chinese, Caucasian and Iranian families of congenital cataracts. Further functional studies are needed to confirm the variant pathogenicity.MethodsThe purpose of this study is to identify the genetic causes that contribute to congenital cataracts with esotropia and nystagmus in a Chinese family. Whole-exome sequencing was performed on samples from all five family members. The two brothers of the father and their daughters were then enrolled in the study, and 40 suspected variants were sequenced among the 9 subjects using Sanger sequencing. The mRNA and protein levels of CRYBA1 in the lens epithelium from cataract patients and normal controls were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. The wild-type and mutated forms (p.G91del) of CRYBA1 cDNA were transfected into two types of cell lines, and the expression level of exogenous CRYBA1 was measured by Western blot analysis. The exogenous CRYBA1 proteins were visualized by immunofluorescence staining.ResultsIn this two-generation family, all three descendants inherited congenital cataracts with esotropia and nystagmus from the father, while the mother’s lens was normal. After two rounds of sequencing, CRYBA1 (c. 269–271 del, p.G91del) was identified as the mutation responsible for the autosomal dominant congenital cataract in the Chinese family. CRYBA1 showed lower expression in cataract lenses than in control lenses. The deleted form (p.G91del) of CRYBA1 showed lower expression and was more aggregate to the cell membrane than the wild-type CRYBA1.ConclusionsWe performed molecular experiments to confirm that the p.G91del mutation in CRYBA1 results in abnormal expression and distribution of CRYBA1 protein, and this study could serve as an example of the pathogenicity of an in-frame small deletion in an inherited eye disorder.

Abstract 505: Adenovirus Targets Connexin43 Gap Junction Expression and Function During Infection

Introduction: Myocarditis leads to pathologic remodeling of the myocardium and arrhythmia. A leading etiological agent of viral myocarditis is human Adenovirus type 5 (Ad5). Individuals affected are at a higher risk of arrhythmogenesis which can lead to sudden cardiac death. Gap junctions, comprised of connexin proteins, provide direct intercellular communication between cardiomyocytes to facilitate electrical coupling. Connexin43 (Cx43) is the predominant connexin expressed in the working myocardium and is tightly regulated by post-translational modifications. Gap junctions are also known to propagate the innate antiviral immune response but the impact of Ad5 infection on Cx43 expression and function is essentially unexplored. Hypothesis: During Ad5 infection Cx43 gap junctions are directly targeted to limit intercellular communication thus precipitating an arrhythmogenic substrate. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), keratinocytes (HaCaT), and lentivirus-transduced HaCaTs stably overexpressing Cx43 were infected with Ad5. Biochemical fractionation and pulse chase assays were implemented to determine gap junction and Cx43 stability. Cx43 protein levels and phosphorylation status were determined by western blotting and immunoprecipitation experiments. Confocal immunofluorescence microscopy was employed determine localization and functional of Cx43 during Ad5 infection. Results: Cx43 expression is targeted early in infection with remaining protein localized at the cell-cell boarder during infection. Phosphorylation of Cx43-Ser368 was detected during early Ad5 infection implicating reduced gap junction conductance. In contrast phosphorylation of Cx43-Ser262 is reduced during Ad5 infection with a concomitant increase in protein half-life despite losses in functional gap junction intercellular communication. Conclusion: Cx43 gap junction structures are stabilized during Ad5 infection but directly targeted by specific phosphorylation events to effect channel closure. Insight provided into mechanisms by which Ad5 manipulates Cx43 regulation will provide novel therapeutic avenues to target gap junction function in a broad spectrum of heart disease states.

Calcium-calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions.

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

Connexin43 Gap Junctions and the Control of Skeletal Remodeling

Osteoblasts, osteoprogenitors, and osteocytes share and amplify signals through connexin43‐containing gap junctions, coordinating their function in response to local and systemic cues. Thus, connexin43 helps orchestrate bone formation and remodeling to maintain cortical bone integrity. Loss or defects in connexin43 lead to alterations in cortical bone quality and skeletal dysplasia. Using in vitro and in vivo models, our data show how connexin43 regulates the differentiation and activity of osteoblasts and development of skeletal elements through the control of signaling pathways that converge on Runx2, a transcriptional regulator of the osteoblast lineage. In addition, our data show how connexin43 can regulate signal transduction cascades by recruiting signaling complexes to the connexin43 C‐terminus, and how loss of this function can recapitulate many of the skeletal phenotypes caused by loss of connexin43 protein in bone. Work with chimeric connexins composed of connexin43 and connexin45, two connexins with opposing biological functions in bone, underscores that both the connexin channel (e.g., pore function) and connexin C‐terminal tail (e.g., signal complexing docking function) act in concert to regulate downstream signaling. Finally, our preliminary data shows that communication of the second messenger, cAMP, through connexin43 gap junctions spatially coordinates bone formation by linking cell populations across surprisingly large distances.Support or Funding InformationThis work was supported by a grant, R01‐AR063631 (JPS), from the National Institutes of Health/National Institute for Arthritis, Musculoskeletal and Skin Diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Connexin Gap Junction Proteins in Development and Disease

Connexins form tightly‐regulated large‐pore channels that facilitate the passage of various ions, metabolites, and signaling molecules directly between cells, or in some cases and/or pathologies, to the extracellular milieu. The human connexin encoding gene family consists of 21 members and mutations in half of these genes leads to over two dozen diseases ranging in severity from manageable developmental abnormalities to life‐shortening organ failure. Developmentally, connexin expression and gap junctional intercellular communication begins at the 8 cell stage and persists throughout organogenesis resulting in most adult cells expressing two or more connexin subtypes. Connexin genes are exquisitely regulated during all developmental processes before birth, and in many tissues like the skin, after birth. Connexins regulate their function by channel gating processes and notably through their turnover owing to their short half‐life of only a few hours. Given their ubiquitous distribution it is not at all surprising that gene mutations lead to disease. In fact, it is probably more surprising that patients harbouring connexin gene mutations do not present with more clinical morbidities. Our laboratory has used tissue‐relevant cells, primary cells, organotypic cultures, mouse models of human diseases, and induced pluripotent stem cells from connexin‐linked disease patients to uncover ten distinct mechanisms by which connexin gene mutations cause disease. Collectively, connexin‐disease linked mutants have been classified into distinct gain‐ and loss‐of function molecular mechanisms of action. Our long‐term goals include developing strategies to compensate or overcome the cellular and tissue defects triggered by these mutants. This presentation will discuss how connexins have emerged as therapeutic targets in disease and in injury repair. We anticipate that once it is better understood how connexin gene mutations cause disease and abnormalities, which often present more acutely during aging, these findings could be translated into pre‐clinical studies leading to possible treatments for gap junction‐linked diseases.Support or Funding InformationSupported by the Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impairment of pericyte-endothelium crosstalk leads to blood-brain barrier dysfunction following traumatic brain injury

The blood-brain barrier (BBB) constitutes a neurovascular unit formed by microvascular endothelial cells, pericytes, and astrocytes. Brain pericytes are important regulators of BBB integrity, permeability, and blood flow. Pericyte loss has been implicated in injury; however, how the crosstalk among pericytes, endothelial cells, and astrocytes ultimately leads to BBB dysfunction in traumatic brain injury (TBI) remains elusive. In this study, we demonstrate the importance of pericyte-endothelium interaction in maintaining the BBB function. TBI causes the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β signaling impairment that results in loss of interaction with endothelium and leads to neurovascular dysfunction. Using in vivo mild (7 psi) and moderate (15 psi) fluid percussion injury (FPI) in mice, we demonstrate the expression of various pericyte markers including PDGFR-β, NG2 and CD13 that were significantly reduced with a subsequent reduction in the expression of various integrins; adherent junction protein, N-cadherin; gap junction protein, connexin-43; and tight junction proteins such as occludin, claudin-5, ZO-1, and JAM-a. Impairment of pericyte-endothelium interaction increases the BBB permeability to water that is marked by a significant increase in aquaporin4 expression in injured animals. Similarly, pericyte-endothelium integrity impairment in FPI animals greatly increases the permeability of small-molecular-weight sodium fluorescein and high-molecular-weight-tracer Evans blue across the BBB. In addition, the injury-inflicted animals show significantly higher levels of S100β and NSE in the blood samples compared with controls. In conclusion, our data provide an insight that brain trauma causes an early impairment of pericyte-endothelium integrity and results in BBB dysregulation that initiates pathological consequences associated with TBI.

The involvement of the ERK-MAPK pathway in TGF-β1–mediated connexin43-gap junction formation in chondrocytes

ABSTRACTPurposes: Gap junction intercellular communication (GJIC) exhibits a key role in maintaining the homeostasis of articular cartilage. Connexin43 (Cx43) protein is predominant in the structures that form gap junctions. We aim to determine the potential underlying mechanisms of TGF-β1 (Transforming growth factor-β1)—regulated cell communication in chondrocytes.Materials and methods: After exposure of chondrocytes to recombinant TGF-β1, quantitative real-time PCR was used to detect expression levels of Cx43 mRNA. Western blot analysis was used to check Cx43 and mitogen-activated protein kinase (MAPK) family components. Immunofluorescence staining was performed to confirm ERK-MAPK pathway activation and Cx43 protein distribution. MAPK inhibitors (ERK inhibitor U0126, JNK inhibitor SP 600125 and P38 inhibitor SP 203580) were applied to verify the specificity effects of ERK-MAPK pathway. GJIC between chondrocytes were evaluated using Scrape loading/dye transfer (SLDT) assay.Results: It was first found that TGF-β1modulatedthe Cx43protein expressions and its sub-cellular distribution. TGF-β1 promoted gap junction intercellular communication (GJIC) formations in chondrocytes, especially in a higher cell intensity. ERK-MAPK signaling pathway was activated in TGF-β1-mediated gap junctions among chondrocytes. Furthermore, the inhibitor of ERK attenuated the increases of Cx43 expressions and functional gap junction formations induced by TGF-β1, while cross-talk between ERK-MAPK and Smad signal pathways exists shown in the process.Conclusions: This study provides evidence to show the importance of the ERK-MAPK pathway in TGF-β1—mediated Cx43 expression and functional gap junction formation.

Are there gap junctions without connexins or pannexins?

BackgroundGap junctions (GJ) are one of the most common forms of intercellular communication. GJs are assembled from proteins that form channels connecting the cytoplasm of adjacent cells. They are considered to be the main or the only type of intercellular channels and the universal feature of all multicellular animals. Two unrelated protein families are currently considered to be involved in this function, namely, connexins and pannexins (pannexins/innexins). Pannexins were hypothesized to be the universal GJ proteins of multicellular animals, distinct from connexins that are characteristic of chordates only. Here we have revised this supposition by applying growing high throughput sequencing data from diverse metazoan species.ResultsPannexins were found in Chordates, Ctenophores, Cnidarians, and in the most major groups of bilateral protostomes. Yet some metazoans appear to have neither connexins nor pannexins in their genomes. We detected no connexins or pannexins/innexins homologues in representatives of all five classes of echinoderms and their closest relatives hemichordates with available genomic sequences. Despite this, our intracellular recordings demonstrate direct electrical coupling between blastomeres at the 2-cell embryo of the echinoderm (starfish Asterias rubens). In these experiments, carboxyfluorescein fluorescent dye did not diffuse between electrically coupled cells. This excludes the possibility that the observed electrical coupling is mediated by incomplete cytoplasm separation during cleavage.ConclusionFunctional GJs are present in representatives of the clade that lack currently recognized GJ protein families. New undiscovered protein families utilized for intercellular channels are predicted. It is possible that the new type(s) of intercellular channels are present in parallel to pannexin and connexin gap junctions in animal groups, other than Echinodermata.

DACT1 Involvement in the Cytoskeletal Arrangement of Cardiomyocytes in Atrial Fibrillation by Regulating Cx43.

ObjectiveTo determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF).MethodsThe DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson’s staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting.ResultsDecreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman’s rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman’s rho=0.370) and changed the Cx43 distribution in cardiac cell lines.ConclusionDACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Connexin-43 Gap Junctions Are Responsible for the Hypothalamic Tanycyte-Coupled Network.

Tanycytes are hypothalamic radial glia-like cells that form the basal wall of the third ventricle (3V) where they sense glucose and modulate neighboring neuronal activity to control feeding behavior. This role requires the coupling of hypothalamic cells since transient decreased hypothalamic Cx43 expression inhibits the increase of brain glucose-induced insulin secretion. Tanycytes have been postulated as possible hypothalamic neuronal precursors due to their privileged position in the hypothalamus that allows them to detect mitogenic signals and because they share the markers and characteristics of neuronal precursors located in other neurogenic niches, including the formation of coupled networks through connexins. Using wild-type (WT), Cx30−/– and Cx30−/–, Cx43fl/fl:glial fibrillary acidic protein (GFAP)-Cre (double knockout, dKO) mouse lines, we demonstrated that tanycytes are highly coupled to each other and also give rise to a panglial network specifically through Cx43. Using the human GFAP (hGFAP)-enhanced green fluorescent protein (EGFP) transgenic mouse line, we provided evidence that the main parenchymal-coupled cells were astrocytes. In addition, electrophysiological parameters, such as membrane resistance, were altered when Cx43 was genetically absent or pharmacologically inhibited. Finally, in the dKO mouse line, we detected a significant decrease in the number of hypothalamic proliferative parenchymal cells. Our results demonstrate the importance of Cx43 in tanycyte homotypic and panglial coupling and show that Cx43 function influences the proliferative potential of hypothalamic cells.

Probing Endothelial Cell Mechanics Through Connexin 43 Disruption.

The endothelium has been established to generate intercellular stresses and suggested to transmit these intercellular stresses through cell-cell junctions, such as VE-Cadherin and ZO-1, for example. Although the previously mentioned molecules reflect the appreciable contributions both adherens junctions and tight junctions are believed to have in endothelial cell intercellular stresses, in doing so they also reveal the obscure relationship that exists between gap junctions and intercellular stresses. Therefore, to bring clarity to this relationship we disrupted expression of the endothelial gap junction connexin 43 (Cx43) by exposing confluent human umbilical vein endothelial cells (HUVECs) to a low (0.2 μg/mL) and high (2 μg/mL) concentration of 2,5-dihydroxychalcone (chalcone), a known Cx43 inhibitor. To evaluate the impact Cx43 disruption had on endothelial cell mechanics we utilized traction force microscopy and monolayer stress microscopy to measure cell-substrate tractions and cell-cell intercellular stresses, respectively. HUVEC monolayers exposed to a low concentration of chalcone produced average normal intercellular stresses that were on average 17% higher relative to control, while exposure to a high concentration of chalcone yielded average normal intercellular stresses that were on average 55% lower when compared to control HUVEC monolayers. HUVEC maximum shear intercellular stresses were observed to decrease by 16% (low chalcone concentration) and 66% (high chalcone concentration), while tractions exhibited an almost 2-fold decrease under high chalcone concentration. In addition, monolayer cell velocities were observed to decrease by 19% and 35% at low chalcone and high chalcone concentrations, respectively. Strain energies were also observed to decrease by 32% and 85% at low and high concentration of chalcone treatment, respectively, when compared to control. The findings we present here reveal for the first time the contribution Cx43 has to endothelial biomechanics.

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Abstract 16931: Alternate UTR Usage Regulates Connexin43 mRNA Translation Limiting Gap Junction Formation During Stress

Introduction: Connexin43 (Cx43, Gja1 ) gap junctions enable electrical coupling of cardiomyocytes necessary for heart function. Pathological disruption of Cx43 expression or localization results in the arrhythmias of sudden cardiac death. Investigation of Cx43 gap junction biology has focused on transcription, vesicular trafficking, and post translational modifications. The importance of translational regulation however is highlighted by the recent discovery that Cx43 mRNA undergoes internal translation initiation to yield truncated protein isoforms, including the predominant GJA1-20k isoform capable of regulating gap junction formation. We have found that TGF-β signaling, a hallmark of cardiac stressors including hypertrophy, suppresses GJA1-20k expression to limit gap junction formation. The mechanisms regulating Cx43 mRNA internal translation in response to cellular stress are unknown but are critical in harnessing this biology for therapeutic restoration of electrical coupling in diseased hearts. We hypothesize that altered Gja1 -5’UTR usage promotes translation of full length Cx43 over internal translation products resulting in reduced gap junction formation during stress. Methods: Rapid amplification of cDNA ends was used to identify Gja1- 5’UTR isoforms following TGF-β exposure or hypoxia, and Western blotting was used to monitor Gja1 translation. Identified Gja1 -5’UTR variants were transfected into Cx43 knockout cells to assess translational potential. Luciferase assays assessed potential IRES activity within Gja1 mRNA in relation to specific 5’UTR isoforms. Results: TGF-β or hypoxia significantly increase translation of full length Cx43 over Gja1-20k correlating with an increase in truncated Gja1 -5’UTRs. Sequenced UTR isoforms map to genome wide transcription start sites and transfection of Gja1 transcripts with truncated 5’UTRs is sufficient to reduce Gja1-20k translation, phenocopying stress models. Conclusions: Alteration of Gja1 -5’UTR composition represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation to fine tune intercellular communication during stress.

Site specific hypermethylation of CpGs in Connexin genes 30, 26 and 43 in different grades of glioma and attenuated levels of their mRNAs

Aim: Gliomas, the intracranial tumours are considered the deadliest malignancies. The gap junctional Connexins (Cxs) that maintain cellular homeostasis perform a unique function in glial tumour suppression. However, the differential methylation patterns of Cxs were not revealed in glioma so far. The current study attempts to categorise promoter methylation of Cx30 and Cx26 and intron methylation of Cx43 in different grades of human glioma.Materials and methods: About 85 glioma patients with pathologically confirmed grades and 15 control brain tissues were recruited in the study. Bisulphite-PCR-Single Stranded Conformation analysis(SSCA), Bisulphite sequencing and MeDIP-qPCR were carried out to assess methylation status and Cx mRNA levels were also analysed to evaluate the effect of methylation.Results: We found that promoter CpG islands(CpGs) reside in Sp1 and Ap2 sites of Cx30 and 26 were hypermethylated in high grades (HG) of glioma rather than low grades. The input % of both was significantly increased (p < 0.03) in progressive grades. Interestingly, Cx43 could exhibit a significant increase (p < 0.05) in input % only in grade IV. While, Cx30 and 26 mRNAs were downregulated according to their methylation status in progressive fashion with grades, Cx43 was downregulated irrespective of intron methylation.Conclusion: Thus, we suggest that the sites and extent of methylation of Cxs (30 and 26 but not in 43) are found to be altered. In different grades of glioma can provide better appreciation of the grade of the patient and might help in strategies based on epigenetic approaches.

Directional coupling of oligodendrocyte connexin-47 and astrocyte connexin-43 gap junctions.

Intercellular communication via gap junction channels between oligodendrocytes and between astrocytes as well as between these cell types is essential to maintain the integrity of myelin in the central nervous system. Oligodendrocyte gap junction connexin-47 (Cx47) is a key element in this crosstalk and indeed, mutations in human Cx47 cause severe myelin disorders. However, the permeation properties of channels of Cx47 alone and in heterotypic combination with astrocyte Cx43 remain unclear. We show here that Cx47 contains three extra residues at 5' amino-terminus that play a critical role in the channel pore structure and account for relative low ionic conductivity, cationic permselectivity and voltage-gating properties of oligodendrocyte-oligodendrocyte Cx47 channels. Regarding oligodendrocyte-astrocyte coupling, heterotypic channels formed by Cx47 with Cx43 exhibit ionic and chemical rectification, which creates a directional diffusion barrier for the movement of ions and larger negatively charged molecules from cells expressing Cx47 to those with Cx43. The restrictive permeability of Cx47 channels and the diffusion barrier of Cx47-Cx43 channels was abolished by a mutation associated with leukodystrophy, the Cx47P90S, suggesting a novel pathogenic mechanism underlying myelin disorders that involves alterations in the panglial permeation.

Differences in Functional Expression of Connexin43 and NaV1.5 by Pan- and Class-Selective Histone Deacetylase Inhibition in Heart.

Class-selective histone deacetylase (HDAC) inhibitors were designed to improve safety profiles and therapeutic effectiveness in the treatment of multiple cancers relative to pan-HDAC inhibitors. However, the underlying mechanisms for their therapeutic and cardiotoxic potentials remain poorly understood. Cardiac sodium currents (INa) and gap junction conductance (gj) were measured by whole cell patch clamp techniques on primary cultures of neonatal cardiomyocytes. Cardiac NaV1.5 sodium channel and connexin43 (Cx43) gap junction protein levels were assessed by Western blot analyses. Panobinostat produced concentration-dependent reductions in ventricular gj, peak INa density, and NaV1.5 protein expression levels. Membrane voltage (Vm)-dependent activation of INa was shifted by +3 to 6 mV with no effect on inactivation. Entinostat (1 μM) did not affect ventricular gj, peak INa density, or INa activation. However, the INa half-inactivation voltage (V½) was shifted by −3.5 mV. Ricolinostat had only minor effects on ventricular gj and INa properties, though INa activation was shifted by −4 mV. Cx43 and NaV1.5 protein expression levels were not altered by class-selective HDAC inhibitors. The lack of effects of class-selective HDAC inhibitors on ventricular gj and INa may help explain the improved cardiac safety profile of entinostat and ricolinostat.

Intrinsic Oncogenic Function of Intracellular Connexin26 Protein in Head and Neck Squamous Cell Carcinoma Cells.

It has long been known that the gap junction is down-regulated in many tumours. One of the downregulation mechanisms is the translocation of connexin, a gap junction protein, from cell membrane into cytoplasm, nucleus, or Golgi apparatus. Interestingly, as tumours progress and reinforce their malignant phenotype, the amount of aberrantly-localised connexin increases in different malignant tumours including oesophageal squamous cell carcinoma, thus suggesting that such an aberrantly-localised connexin should be oncogenic, although gap junctional connexins are often tumour-suppressive. To define the dual roles of connexin in head and neck squamous cell carcinoma (HNSCC), we introduced the wild-type connexin26 (wtCx26) or the mutant Cx26 (icCx26) gene, the product of which carries the amino acid sequence AKKFF, an endoplasmic reticulum-Golgi retention signal, at the C-terminus and is not sorted to cell membrane, into the human FaDu hypopharyngeal cancer cell line that had severely impaired the expression of connexin during carcinogenesis. wtCx26 protein was trafficked to the cell membrane and formed gap junction, which successfully exerted cell-cell communication. On the other hand, the icCx26 protein was co-localised with a Golgi marker, as revealed by immunofluorescence, and thus was retained on the way to the cell membrane. While the forced expression of wtCx26 suppressed both cell proliferation in vitro and tumorigenicity in mice in vivo, icCx26 significantly enhanced both cell proliferation and tumorigenicity compared with the mock control clones, indicating that an excessive accumulation of connexin protein in intracellular domains should be involved in cancer progression and that restoration of proper subcellular sorting of connexin might be a therapeutic strategy to control HNSCC.

Effects of Pinocembrin Pretreatment on Connexin 43 (Cx43) Protein Expression After Rat Myocardial Ischemia-Reperfusion and Cardiac Arrhythmia.

BackgroundCardiac infarction frequently leads to arrhythmia and ischemia/reperfusion (I/R) aggravates cardiac injury. Pinocembrin can resist cerebral ischemia and decrease cardiac infarction area. This study thus generated a rat myocardial I/R model to assess the effect on ventricular rhythm and expression of gap junction connexin (Cx43).Material/MethodsMale SD rats were randomly assigned into sham, model, and pinocembrin (30 mg/kg) pretreatment groups (N=15 each). The I/R model was generated by ligation of the left anterior descending coronary artery for 30 min. The pinocembrin group received intravenous injection 10 min before surgery. Heart rate (HR), mean artery pressure (MAP), rate pressure product (RPP), and arrhythmia were observed at 10 min before ischemia, 30 min after ischemia, and at 30, 60, and 120 min after reperfusion. ELISA was used to assess serum CK-MB and cTnI levels. Na+-K+ATPase and Ca+-Mg2+ATPase levels were quantified by spectrometry, followed by HE staining, IHC approach for Cx43 expression, and Western blot for Kir2.1 protein expression.ResultsModel rats had significantly lower HR, MAP, and RPP than in the sham group, and the pinocembrin pretreatment group had higher serum indexes. Arrhythmia index, CK-MB, and cTnI were higher in the model and pinocembrin groups, while Na+-K+ATPase, Ca+-Mg2+ATPase, Cx43, and Kir2.1 proteins were lower (p<0.05).ConclusionsPinocembrin alleviated ventricular arrhythmia in I/R rats via enhancing Na+-K+ATPase and Ca+-Mg2+ATPase activity and upregulating Cx43 and Kir2.1 protein expression.