Modulation Of Gap Junctions Research Articles

Robotic manipulation of cardiomyocytes to identify gap junction modifiers for arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 (PKP2). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells' contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction-mediated diseases.

Abstract Mo068: The role of FGF13 in modulating gap junction protein Connexin-43

Background: Fibroblast growth factor homologous factors (FHFs) are associated with arrhythmias like Brugada syndrome and atrial fibrillation, but their pathological mechanisms remain poorly understood. FHFs are best characterized as regulators of voltage gated sodium channels (VGSCs), however recent studies suggest broader, non-VGSC-related functions, including regulation of microtubules. Whether and how non-VGSC regulatory functions modulate cardiac rhythm is not known. Hypothesis: FGF13 regulates Cx43 trafficking and expression in ventricular cardiomyocytes via destabilizing microtubules. Aims: Building on an unbiased proteomic screen, we investigated if FGF13, the predominant FHF in rodent heart, affects the expression and trafficking of Connexin-43 (Cx43), the predominant gap junction protein in ventricular myocytes. Methods: To identify additional FGF13-dependent functional roles, we employed proximity labeling proteomics in mouse heart with the TurboID biotin ligase fused to FGF13. We used immunoblots, immunohistochemistry, and immunocytochemistry to further characterize the effects of FGF13 ablation on Cx43 pattern distribution and expression. We also leveraged electrophysiology techniques using gap junction inhibitors to detect any Cx43-dependent functional changes in FGF13-ablated cardiomyocytes. Results: The proteomic data revealed that cell junction proteins associated with the intercalated disc (ID) were among the top FGF13 near neighbors, of which Cx43 was heavily enriched, together with microtubule associated proteins (MAPs) known to be critical for cardiomyocyte function and ion channel trafficking. In ventricular cardiomyocytes from constitutive cardiomyocyte-restricted Fgf13 knockout mice, we found markedly perturbed trafficking of Cx43 and reduced colocalization with the ID marker N-Cadherin. Separately, MAP4 abundance was markedly reduced. The addition of colchicine, a microtubule destabilizing drug, to WT cells altered Cx43 distribution pattern similar to that observed in FGF13-ablated cardiomyocytes. Re-expression of FGF13 restored Cx43 trafficking and ID targeting. Electrophysiology experiments using a Cx43 hemichannel inhibitor, Gap19, showed a perturbation of the action potential duration in knockout cardiomyocytes. Isoproterenol-induced action potential prolongation in knockout myocytes was decreased by the addition of Gap19. Conclusions: FGF13 regulates microtubule-based trafficking and targeting of Cx43.

The Impact of Chronic Magnesium Deficiency on Excitable Tissues-Translational Aspects.

Neuromuscular excitability is a vital body function, and Mg2+ is an essential regulatory cation for the function of excitable membranes. Loss of Mg2+ homeostasis disturbs fluxes of other cations across cell membranes, leading to pathophysiological electrogenesis, which can eventually cause vital threat to the patient. Chronic subclinical Mg2+ deficiency is an increasingly prevalent condition in the general population. It is associated with an elevated risk of cardiovascular, respiratory and neurological conditions and an increased mortality. Magnesium favours bronchodilation (by antagonizing Ca2+ channels on airway smooth muscle and inhibiting the release of endogenous bronchoconstrictors). Magnesium exerts antihypertensive effects by reducing peripheral vascular resistance (increasing endothelial NO and PgI2 release and inhibiting Ca2+ influx into vascular smooth muscle). Magnesium deficiency disturbs heart impulse generation and propagation by prolonging cell depolarization (due to Na+/K+ pump and Kir channel dysfunction) and dysregulating cardiac gap junctions, causing arrhythmias, while prolonged diastolic Ca2+ release (through leaky RyRs) disturbs cardiac excitation-contraction coupling, compromising diastolic relaxation and systolic contraction. In the brain, Mg2+ regulates the function of ion channels and neurotransmitters (blocks voltage-gated Ca2+ channel-mediated transmitter release, antagonizes NMDARs, activates GABAARs, suppresses nAChR ion current and modulates gap junction channels) and blocks ACh release at neuromuscular junctions. Magnesium exerts multiple therapeutic neuroactive effects (antiepileptic, antimigraine, analgesic, neuroprotective, antidepressant, anxiolytic, etc.). This review focuses on the effects of Mg2+ on excitable tissues in health and disease. As a natural membrane stabilizer, Mg2+ opposes the development of many conditions of hyperexcitability. Its beneficial recompensation and supplementation help treat hyperexcitability and should therefore be considered wherever needed.

Integrating and optimizing tonabersat in standard glioblastoma therapy: A preclinical study.

Glioblastoma (GB), a highly aggressive primary brain tumor, presents a poor prognosis despite the current standard therapy, including radiotherapy and temozolomide (TMZ) chemotherapy. Tumor microtubes involving connexin 43 (Cx43) contribute to glioma progression and therapy resistance, suggesting Cx43 inhibition as a potential treatment strategy. This research aims to explore the adjuvant potential of tonabersat, a Cx43 gap junction modulator and blood-brain barrier-penetrating compound, in combination with the standard of care for GB. In addition, different administration schedules and timings to optimize tonabersat's therapeutic window are investigated. The F98 Fischer rat model will be utilized to investigate tonabersat's impact in a clinically relevant setting, by incorporating fractionated radiotherapy (three fractions of 9 Gy) and TMZ chemotherapy (29 mg/kg). This study will evaluate tonabersat's impact on tumor growth, survival, and treatment response through advanced imaging (CE T1-w MRI) and histological analysis. Results show extended survival in rats receiving tonabersat with standard care, highlighting its adjuvant potential. Daily tonabersat administration, both preceding and following radiotherapy, emerges as a promising approach for maximizing survival outcomes. The study suggests tonabersat's potential to reduce tumor invasiveness, providing a new avenue for GB treatment. In conclusion, this preclinical investigation highlights tonabersat's potential as an effective adjuvant treatment for GB, and its established safety profile from clinical trials in migraine treatment presents a promising foundation for further exploration.

Gap junction modulators: Prospects in bupropion cardiotoxicity

Bupropion, a multifaceted antidepressant, is associated with serious cardiotoxicity when misused. Recently, there has been a noticeable increase in cases of intentional poisoning and current therapeutic interventions for bupropion overdose remain insufficient. The cardiotoxic effects of the drug are primarily linked to its influence on cardiac gap junctions. Small-molecule drugs enhancing gap junction conductance, such as rotigaptide and danegaptide, present potential as antidotal agents. Testing these molecules under conditions of bupropion overdose could address the urgent need for innovative approaches to mitigate bupropion-induced cardiotoxicity.

The rectification of heterotypic Cx46/Cx50 gap junction channels depends on intracellular magnesium.

Gap junction (GJ) intercellular communication is crucial in many physiological and pathological processes. A GJ channel is formed by head-to-head docking of two hexameric hemichannels from two neighboring cells. Heterotypic GJ channels formed by two different homomeric connexin hemichannels often display rectification properties in the current-voltage relationship while the underlying mechanisms are not fully clear. Here we studied heterotypic Cx46/Cx50 GJs at a single GJ channel level. Our data showed unitary Cx46/Cx50 GJ channel conductance (γj) rectification when 5 mmol/L Mg2+ was included in the patch pipette solution, while no γj rectification was observed when no Mg2+ was added. Including 5 mmol/L Mg2+ in pipette solution significantly decreased the γj of homotypic Cx46 GJ with little change in homotypic Cx50 γj. A missense point variant in Cx46 (E43F) reduced the Mg2+-dependent reduction in γj of Cx46 E43F GJ, indicating that E43 might be partially responsible for Mg2+-dependent decrease in γj of Cx46. A comprehensive understanding of Mg2+ modulation of GJ at the individual channel level is useful in understanding factors in modulating GJ-mediated intercellular communication in health and diseases.

Restoring connexin-36 function in diabetogenic environments precludes mouse and human islet dysfunction.

The secretion of insulin from β-cells in the islet of Langerhans is governed by a series of metabolic and electrical events, which can fail during the progression of type 2 diabetes (T2D). β-cells are electrically coupled via connexin-36 (Cx36) gap junction channels, which coordinates the pulsatile dynamics of [Ca2+ ] and insulin release across the islet. Factors such as pro-inflammatory cytokines and free fatty acids disrupt gap junction coupling under in vitro conditions. Here we test whether gap junction coupling and coordinated [Ca2+ ] dynamics are disrupted in T2D, and whether recovery of gap junction coupling can recover islet function. We examine islets from donors with T2D, from db/db mice, and islets treated with pro-inflammatory cytokines (TNF-α, IL-1β, IFN-ɣ) or free fatty acids (palmitate). We modulate gap junction coupling using Cx36 over-expression or pharmacological activation via modafinil. We also develop a peptide mimetic (S293) of the c-terminal regulatory site of Cx36 designed to compete against its phosphorylation. Cx36 gap junction permeability and [Ca2+ ] dynamics were disrupted in islets from both human donors with T2D and db/db mice, and in islets treated with pro-inflammatory cytokines or palmitate. Cx36 over-expression, modafinil treatment and S293 peptide all enhanced Cx36 gap junction coupling and protected against declines in coordinated [Ca2+ ] dynamics. Cx36 over-expression and S293 peptide also reduced apoptosis induced by pro-inflammatory cytokines. Critically, S293 peptide rescued gap junction coupling and [Ca2+ ] dynamics in islets from both db/db mice and a sub-set of T2D donors. Thus, recovering or enhancing Cx36 gap junction coupling can improve islet function in diabetes. KEY POINTS: Connexin-36 (Cx36) gap junction permeability and associated coordination of [Ca2+ ] dynamics is diminished in human type 2 diabetes (T2D) and mouse models of T2D. Enhancing Cx36 gap junction permeability protects against disruptions to the coordination of [Ca2+ ] dynamics. A novel peptide mimetic of the Cx36 c-terminal regulatory region protects against declines in Cx36 gap junction permeability. Pharmacological elevation in Cx36 or Cx36 peptide mimetic recovers [Ca2+ ] dynamics and glucose-stimulated insulin secretion in human T2D and mouse models of T2D.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Epicardial Adipose Tissue and Cardiac Arrhythmias

Obesity is associated with an increased risk of atrial and ventricular arrhythmias, including life-threatening ones. Epicardial adipose tissue (EAT) is located deep under the visceral pericardium (epicardium) and is therefore in direct contact with the underlying myocardium. In pathological conditions, EAT undergoes a phenotypic transition from a neighbor with protective properties to a substrate that secretes many substances that change the electrophysiology of cardiomyocytes by modulating ion currents that disrupt intercellular electrical connections and stimulate fibrosis. An excess of EAT can cause atrial and ventricular conduction disturbances, which are already evident with standard electrocardiography, predispose to the occurrence of the re-entry phenomenon and cardiac arrhythmias. Among the mechanisms of arrhythmogenesis under the influence of EAT, modulation of ion channels and gap junctions, fibrous remodeling and fatty infiltration are more often considered. However, most of these mechanisms have been studied in experimental studies and cannot easily be extrapolated to humans. There is convincing evidence of a direct relationship between EAT volume and the severity of atrial fibrillation, as well as the clinical benefit obtained from weight loss in patients with this arrhythmia. It is likely that the benefits of weight loss may extend to ventricular arrhythmias.

Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis.

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acidwere used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conductedon the nanopatterns. Cohesion and GJIC were evaluatedin cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

Methylselenocysteine Potentiates Etoposide-Induced Cytotoxicity by Enhancing Gap Junction Activity.

Homomeric or heteromeric connexin (Cx) hemichannels-composed gap junction (GJ) intercellular channel can mediate direct cell-to-cell communication. Accumulating studies indicate that GJs potentiate the cytotoxicity of antitumor drugs in malignant cells. Methylselenocysteine (MSC), a selenium compound from garlic, has been reported to modulate the activity of antineoplastic drugs, but the underlying mechanism remains unclear. This study investigates the efficacy of MSC on chemotherapeutic drugs-induced cytotoxicity and the relationship between this effect and the regulation of GJ function by MSC. Firstly, a doxycycline-regulated HeLa cell line expressing heteromeric Cx26/Cx32 was used as a tool. Etoposide, but not cisplatin or 5-fluorouracil, showed remarkable cytotoxicity in high-density (with GJ formation) cultures than in low-density (without GJ formation) in transformed HeLa cells. And cell density had no effect on etoposide-mediated cytotoxicity in the absence of Cx expression. MSC substantially enhanced etoposide-induced cytotoxicity, and this effect was only detected in the presence of functional GJs. Subsequently, MSC potentiated structural Cx expression as evidenced by increased dye coupling, but no alteration in Cx mRNA expression level in either transformed or primary cancer cell lines. Finally, a redox mechanism involving glutathione (GSH) was found to be related to the posttranscriptional modulation of Cx expression by MSC in HeLa cells. In conclusion, we provide the novel finding that MSC increases etoposide-mediated cytotoxicity by enhancing GJ activity, due to elevated Cx expression through a GSH-dependent posttranscriptional mechanism. More generally, the study highlights potential benefit of the combination of GJ modulators and chemotherapeutic agents in anticancer treatment.

The Search for Biomarkers and Treatments in Chagas Disease: Insights From TGF-Beta Studies and Immunogenetics.

The anti-inflammatory cytokine transforming growth factor beta (TGF-β) plays an important role in Chagas disease (CD), a potentially life-threatening illness caused by Trypanosoma cruzi. In this review we revisited clinical studies in CD patients combined with in vitro and in vivo experiments, presenting three main sections: an overview of epidemiological, economic, and clinical aspects of CD and the need for new biomarkers and treatment; a brief panorama of TGF-β roles and its intracellular signaling pathways, and an update of what is known about TGF-β and Chagas disease. In in vitro assays, TGF-β increases during T. cruzi infection and modulates heart cells invasion by the parasite fostering its intracellular parasite cycle. TGF-β modulates host immune response and inflammation, increases heart fibrosis, stimulates remodeling, and slows heart conduction via gap junction modulation. TGF-β signaling inhibitors reverts these effects opening a promising therapeutic approach in pre-clinical studies. CD patients with higher TGF-β1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-β as a surrogate biomarker of clinical relevance. Moreover, pre-clinical studies in chronic T. cruzi infected mice proved that inhibition of TGF-β pathway improved several cardiac electric parameters, reversed the loss of connexin-43 enriched intercellular plaques, reduced fibrosis of the cardiac tissue, restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Finally, TGF-β polymorphisms indicate that CD immunogenetics is at the base of this phenomenon. We searched in a Brazilian population five single-nucleotide polymorphisms (-800 G>A rs1800468, -509 C>T rs1800469, +10 T>C rs1800470, +25 G>C rs1800471, and +263 C>T rs1800472), showing that CD patients frequently express the TGF-β1 gene genotypes CT and TT at position -509, as compared to noninfected persons; similar results were observed with genotypes TC and CC at codon +10 of the TGF-β1 gene, leading to the conclusion that 509 C>T and +10 T>C TGF-β1 polymorphisms are associated with Chagas disease susceptibility. Studies in genetically different populations susceptible to CD will help to gather new insights and encourage the use of TGF-β as a CD biomarker.

Investigating oligodendrocyte connexins: Heteromeric interactions between Cx32 and mutant or wild-type forms of Cx47 do not contribute to or modulate gap junction function.

Oligodendrocytes express two gap junction forming connexins, connexin 32 (Cx32) and Cx47; therefore, formation of heteromeric channels containing both Cx47 and Cx32 monomers might occur. Mutations in Cx47 cause both Pelizaeus-Merzbacher-like disease Type 1 (PMLD1) and hereditary spastic paraparesis Type 44 (SPG44) and heteromer formation between these mutants and Cx32 may contribute to the pathogenesis of these disorders. Here, we utilized electrophysiological and antibody-based techniques to examine this possibility. When cells expressing both Cx32 and Cx47 were paired with cells expressing either Cx32 or Cx47, properties were indistinguishable from those produced by cells expressing homotypic Cx32 or Cx47 channels. Similarly, pairing cells expressing both Cx32 and Cx47 with cells expressing Cx30 or Cx43 produced channels indistinguishable from heterotypic Cx32/Cx30 or Cx47/Cx43 channels, respectively. The same assessments were performed on cells expressing Cx32 and four mutant forms of Cx47 (p.I33M associated with SPG44 or p.P87S, p.Y269D or p.M283T associated with PMLD1). None of these mutants showed a functional effect on Cx32. Immunostained cells co-expressing Cx32WT (wild type) and Cx47WT showed a Pearson correlation coefficient close to zero, suggesting that any overlap was due to chance. p.Y269D showed a statistically significant negative correlation with Cx32, suggesting that Cx32 and this mutant overlap less than expected by chance. Co-immunoprecipitation of Cx32 with Cx47WT and mutants show only very low levels of co-immunoprecipitated protein. Overall, our data suggest that interactions between PMLD1 or SPG44 mutants and Cx32 gap junctions do not contribute to the pathogenesis of these disorders.

Danegaptide Prevents TGFβ1-Induced Damage in Human Proximal Tubule Epithelial Cells of the Kidney.

Chronic kidney disease (CKD) is a global health problem associated with a number of comorbidities. Recent evidence implicates increased hemichannel-mediated release of adenosine triphosphate (ATP) in the progression of tubulointerstitial fibrosis, the main underlying pathology of CKD. Here, we evaluate the effect of danegaptide on blocking hemichannel-mediated changes in the expression and function of proteins associated with disease progression in tubular epithelial kidney cells. Primary human proximal tubule epithelial cells (hPTECs) were treated with the beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± danegaptide. qRT-PCR and immunoblotting confirmed mRNA and protein expression, whilst a cytokine antibody array assessed the expression/secretion of proinflammatory and profibrotic cytokines. Carboxyfluorescein dye uptake and ATP biosensing measured hemichannel activity and ATP release, whilst transepithelial electrical resistance was used to assess paracellular permeability. Danegaptide negated carboxyfluorescein dye uptake and ATP release and protected against protein changes associated with tubular injury. Blocking Cx43-mediated ATP release was paralleled by partial restoration of the expression of cell cycle inhibitors, adherens and tight junction proteins and decreased paracellular permeability. Furthermore, danegaptide inhibited TGFβ1-induced changes in the expression and secretion of key adipokines, cytokines, chemokines, growth factors and interleukins. The data suggest that as a gap junction modulator and hemichannel blocker, danegaptide has potential in the future treatment of CKD.

Antiepileptogenic Effect of Retinoic Acid.

Retinoic acid, a metabolite of vitamin A, acts through either genomic or nongenomic actions. The genomic action of retinoids exerts effects on gene transcription through interaction with retinoid receptors such as retinoic acid receptors (RARα, β, and γ) and retinoid X receptors (RXRα, β, and γ) that are primarily concentrated in the amygdala, pre-frontal cortex, and hippocampal areas in the brain. In response to retinoid binding, RAR/RXR heterodimers undergo major conformational changes and orchestrate the transcription of specific gene networks. Previous experimental studies have reported that retinoic acid exerts an antiepileptogenic effect through diverse mechanisms, including the modulation of gap junctions, neurotransmitters, long-term potentiation, calcium channels and some genes. To our knowledge, there are no previous or current clinical trials evaluating the use of retinoic acid for seizure control.

A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

A moderate increase in (55mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. Cx26 hemichannels open in response to moderate elevations of CO2 ( 55mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated of 55mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30mm propionate, we found that acidification in the absence of a change in caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

Synaptic Plasticity After Focal Cerebral Ischemia Was Attenuated by Gap26 but Enhanced by GAP-134.

Objective: Synaptic plasticity is critical for neurorehabilitation after focal cerebral ischemia. Connexin 43 (Cx43), the main component of the gap junction, has been shown to be pivotal for synaptic plasticity. The objective of this study was to investigate the role of the Cx43 inhibitor (Gap26) and gap junction modifier (GAP-134) in neurorehabilitation and to study their contribution to synaptic plasticity after focal ischemia.Methods: Time course expression of both total and phosphorylated Cx43 (p-Cx43) were detected by western blotting at 3, 7, and 14 d after focal ischemia. Gap26 and GAP-134 were administered starting from 3 d post focal ischemia. Neurological performances were evaluated by balance beam walking test and Y-maze test at 1, 3, and 7 d. Golgi staining and transmission electron microscope (TEM) detection were conducted at 7 d for observing dendritic spine numbers and synaptic ultrastructure, respectively. Immunofluorescent staining was used at 7 d for detection of synaptic plasticity markers, including synaptophysin (SYN) and growth-associated protein-43 (GAP-43).Results: Expression levels of both total Cx43 and p-Cx43 were increased after focal cerebral ischemia, peaking at 7 d. Compared with the MCAO group, Gap26 worsened the neurological behavior and decreased the dendritic spine number while GAP-134 improved the neurobehavior and increased the number of dendritic spines. Moreover, Gap26 further destroyed the synaptic structure, concomitant with downregulated SYN and GAP-43, whereas GAP-134 alleviated synaptic destruction and upregulated SYN and GAP-43.Conclusion: These findings suggested that Cx43 or the gap junction was involved in synaptic plasticity, thereby promoting neural recovery after ischemic stroke. Treatments enhancing gap junctions may be potential promising therapeutic measures for neurorehabilitation after ischemic stroke.

NMDAR-mediated modulation of gap junction circuit regulates olfactory learning in C. elegans

Modulation of gap junction-mediated electrical synapses is a common form of neural plasticity. However, the behavioral consequence of the modulation and the underlying molecular cellular mechanisms are not understood. Here, using a C. elegans circuit of interneurons that are connected by gap junctions, we show that modulation of the gap junctions facilitates olfactory learning. Learning experience weakens the gap junctions and induces a repulsive sensory response to the training odorants, which together decouple the responses of the interneurons to the training odorants to generate learned olfactory behavior. The weakening of the gap junctions results from downregulation of the abundance of a gap junction molecule, which is regulated by cell-autonomous function of the worm homologs of a NMDAR subunit and CaMKII. Thus, our findings identify the function of a gap junction modulation in an in vivo model of learning and a conserved regulatory pathway underlying the modulation.

Gap-134, a Connexin43 activator, prevents age-related development of ventricular fibrosis in Scn5a+/− mice

Down-regulation of Connexin43 (Cx43) has often been associated with the development of cardiac fibrosis. We showed previously that Scn5a heterozygous knockout mice (Scn5a+/−), which mimic familial progressive cardiac conduction defect, exhibit an age-dependent decrease of Cx43 expression and phosphorylation concomitantly with activation of TGF-β pathway and fibrosis development in the myocardium between 45 and 60 weeks of age. The aim of this study was to investigate whether Gap-134 prevents Cx43 down-regulation with age and fibrosis development in Scn5a+/− mice.We observed in 60-week-old Scn5a+/− mouse heart a Cx43 expression and localization remodeling correlated with fibrosis. Chronic administration of a potent and selective gap junction modifier, Gap-134 (danegaptide), between 45 and 60 weeks, increased Cx43 expression and phosphorylation on serine 368 and prevented Cx43 delocalization. Furthermore, we found that Gap-134 prevented fibrosis despite the persistence of the conduction defects and the TGF-β canonical pathway activation.In conclusion, the present study demonstrates that the age-dependent decrease of Cx43 expression is involved in the ventricular fibrotic process occurring in Scn5a+/− mice. Finally, our study suggests that gap junction modifier, such as Gap-134, could be an effective anti-fibrotic agent in the context of age-dependent fibrosis in progressive cardiac conduction disease.