Cx43 Gap Junction Research Articles

The Hydrophobic Residues in Amino Terminal Domains of Cx46 and Cx50 Are Important for Their Gap Junction Channel Ion Permeation and Gating.

Lens gap junctions (GJs) formed by Cx46 and Cx50 are important to keep lens transparency. Functional studies on Cx46 and Cx50 GJs showed that the Vj-gating, single channel conductance (γj), gating polarity, and/or channel open stability could be modified by the charged residues in the amino terminal (NT) domain. The role of hydrophobic residues in the NT on GJ properties is not clear. Crystal and cryo-EM GJ structures have been resolved, but the NT domain structure has either not been resolved or has showed very different orientations depending on the component connexins and possibly other experimental conditions, making it difficult to understand the structural basis of the NT in Vj-gating and γj. Here, we generated missense variants in Cx46 and Cx50 NT domains and studied their properties by recombinant expression and dual whole-cell patch clamp experiments on connexin-deficient N2A cells. The NT variants (Cx46 L10I, N13E, A14V, Q15N, and Cx50 I10L, E13N, V14A, N15Q) were all able to form functional GJs with similar coupling%, except Cx46 N13E, which showed a significantly reduced coupling%. The GJs of Cx46 N13E, A14V and Cx50 E13N, N15Q showed a reduced coupling conductance. Vj-gating of all the variant GJs were similar to the corresponding wild-type GJs except Cx46 L10I. The γj of Cx46 N13E, A14V, Cx50 E13N, and N15Q GJs was reduced to 51%, 82%, 87%, and 74%, respectively, as compared to their wild-type γjs. Structural models of Cx46 L10I and A14V predicted steric clashes between these residues and the TM2 residues, which might be partially responsible for our observed changes in GJ properties. To verify the importance of hydrophobic interactions, we generated a variant, Cx50 S89T, which also shows a steric clash and failed to form a functional GJ. Our experimental results and structure models indicate that hydrophobic interactions between the NT and TM2 domain are important for their Vj-gating, γj, and channel open stability in these and possibly other GJs.

Unravelling Contributions of Astrocytic Connexin 43 to the Functional Activity of Brain Neuron-Glial Networks under Hypoxic State In Vitro.

Brain hypoxia remains an Achilles’ heel for public health that must be urgently addressed. Hypoxic damage affects both neurons and glial cells, particularly astrocytes, which are in close dynamic bi-directional communication, and are organized in plastic and tightly regulated networks. However, astroglial networks have received limited attention regarding their influence on the adaptive functional rearrangements of neural networks to oxygen deficiency. Herein, against the background of astrocytic Cx43 gap junction blockade by the selective blocker Gap19, we evaluated the features of spontaneous calcium activity and network characteristics of cells in primary cultures of the cerebral cortex, as well as the expression levels of metabotropic glutamate receptors 2 (mGluR2) and 5 (mGluR5) in the early and late periods after simulated hypoxia in vitro. We showed that, under normoxic conditions, blockade of Cx43 leads to an increase in the expression of metabotropic glutamate receptors mGluR2 and mGluR5 and long-term modulation of spontaneous calcium activity in primary cortical cultures, primarily expressed in the restructuring of the functional architectonics of neuron–glial networks through reducing the level of correlation between cells in the network and the percentage of existing correlated connections between cells. Blocking Cx43 during hypoxic injury has a pronounced neuroprotective effect. Together with the increased expression of mGluR5 receptors, a decrease in mGluR2 expression to the physiological level was found, which suggests the triggering of alternative molecular mechanisms of cell adaptation to hypoxia. Importantly, the blockade of Cx43 in hypoxic damage contributed to the maintenance of both the main parameters of the spontaneous calcium activity of primary cortical cultures and the functional architectonics of neuron–glial networks while maintaining the profile of calcium oscillations and calcium signal communications between cells at a highly correlated level. Our results demonstrate the crucial importance of astrocytic networks in functional brain adaptation to hypoxic damage and could be a promising target for the development of rational anti-hypoxic therapy.

TNF-α Plus IL-1β Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1β increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1β treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1β-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell–cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Modulation of Gap Junction Coupling Within the Islet of Langerhans During the Development of Type 1 Diabetes.

In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca2+ homeostasis. Connexin36 (Cx36) gap junctions electrically couple β-cells to coordinate glucose-stimulated Ca2+ and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca2+ dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6–12 weeks age. Glucose-stimulated insulin secretion, Ca2+ dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and KATP or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca2+ dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by KATP activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca2+. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca2+ homeostasis protects against islet decline in T1D.

The impact of Lactobacillus and Bifidobacterium probiotic cocktail on modulation of gene expression of gap junctions dysregulated by intestinal pathogens.

Probiotics are special bacterial strains with strain specific impacts. They can affect health condition in intestine by producing organic acid, competing with pathogens and maintaining cells homeostasis. Regarding to importance of cell junctions in cells transportation and the influence of pathogens in their functions which lead to inflammation, the impact of probiotic strains comprised of Lactobacillus and Bifidobacterium strains on two important members of gap junctions (Cx26 and Cx43) were assayed. The expressions of cell junction genes in contact with probiotic cocktail along with pathogenic components of enterotoxigenic Escherichia coli and Salmonella typhimurium on HT-29 cell line in different treatment orders were evaluated. Results analysis demonstrated downregulation of cx26 and cx43 along with pathogenic components while, probiotic cocktail could modulate their expression by upregulation. We concluded that Lactobacillus and Bifidobacterium strains were efficient probiotics, when they were used as one cocktail, impacted grater amount on the expression of cell junctions and this might lead to modulate homeostasis and reveal inflammation symptoms in intestine.

Abstract 959: Non-junctional connexin43 complexing with microtubules promotes epithelial-mesenchymal transition

Abstract Epithelial-mesenchymal transition (EMT) is a normal process of embryonic development, stem cell differentiation, and wound healing. During EMT, epithelial cells lose apical-basal polarity, remodel intercellular junctions, and acquire migratory and invasive abilities as they become mesenchymal. Pathologically, EMT has been demonstrated to contribute to intravasation of metastatic tumor cells into the vasculature. Among the activators of EMT, TGF-β has been shown to be most prevalent and is sufficient for experimental induction of EMT. Gap junctions, comprising connexin proteins, allow direct mechanical and metabolic coupling between adjacent cells, and represent a major intercellular structure altered during EMT. Connexin43 (Cx43) is a gap junction protein that has been implicated as both a tumor suppressor and enhancer depending on cancer stage. Our previous work revealed that despite losses in Cx43 gap junctions during EMT, mRNA and cytosolic levels of Cx43 protein increased. Such a shift of Cx43 to cytoplasmic pools has previously been observed in cancer progression and research suggests that non-junctional Cx43 can regulate the cytoskeleton, including microtubule stability. The C-terminus of Cx43 encompasses a tubulin binding domain which, while implicated in Cx43 trafficking, remains functionally poorly understood. We have identified a tumorigenic role for non-junctional Cx43 through its interaction with microtubules in glioma cancer stem cells. This led us to hypothesize that increased cytosolic Cx43 during EMT promotes Cx43-microtubule interaction to facilitate mobility and invasion of metastatic cancer cells. Using several independent TGF-β-induced EMT models, we observe increased complexing of Cx43 and microtubules during EMT by super-resolution microscopy. To study the role of Cx43 interaction with microtubules during EMT, we generated Cx43 harboring mutations in the tubulin binding domain which were ectopically expressed by adenoviral transduction in Cx43 knockout CRISPR/Cas9 epithelial cells. We find these Cx43 mutants fail to interact with microtubules compared to wild-type Cx43. Turning to TGF-β-induced EMT, ablation of the Cx43/microtubules interaction with these mutants is sufficient to decrease expression of mesenchymal markers and inhibit cell migration. We are currently investigating the effects of disrupting Cx43/microtubule complexing in vivo with a breast cancer mouse model to further elucidate effects on tumor progression and metastasis. Given the complex relationship between connexins and cancer progression, our findings support targeting this pathological ‘non-junctional’ Cx43 population specifically to limit metastatic disease, as Cx43 gap junction function is critical to normal tissue function and contributes to tumor suppression. Citation Format: Aryo Sorayya, Christina Wheeler, Stacie Deaver, Kenneth Young, Michael Zeitz, James Smyth, Samy Lamouille. Non-junctional connexin43 complexing with microtubules promotes epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 959.

Abstract 1053: Combination therapy with nonthermal plasma and tirapazamine selectively targets glioblastoma cells and regulates the gap junction phenotype

Abstract Considering the side effects common to most chemotherapeutic agents, a novel approach was developed to selectively treat cancer by targeting the oxidative stress pathway which is induced in many tumor types. Our previous studies demonstrated that nonthermal plasma (NTP) selectively causes apoptotic cell death in melanoma cells while preserving the normal surrounding keratinocytes. The prodrug, tirapazamine, was shown to potentiate the effects of NTP while maintaining selectivity because tirapazamine only activates in hypoxic conditions, an environment common to many tumors. This patented combination therapy has an additive-to-synergistic effect on inducing apoptosis in melanoma. Previous studies have also demonstrated efficacy in a broad range of tumor cells including cervical carcinoma, colon cancer, and pancreatic adenocarcinoma. This report demonstrates for the first time that while NTP alone kills a targeted area of glioblastoma cells and tirapazamine alone kills most of the susceptible cells, the combination of NTP and tirapazamine kills all the cells throughout the tissue culture plate. The mechanism for the increase in the surface area of cell death was identified as the passage of reactive oxygen species (ROS) by intercellular communication through gap junctions. The IC50 dose of tirapazamine required to target several glioblastoma cell lines was less than that for melanoma cells. Through parameter optimization with varied plasma conditions and altered drug dosing, the selective killing of glioblastoma cells was achieved without affecting normal primary astrocytes. Primary astrocytes and glioblastoma cells were co-cultured to observe the effect on the optimal targeting dose of tirapazamine. Glioblastoma cells had an IC50 at hypoxia which was reduced as compared to astrocytes in their normoxia state. The Glioblastoma IC50 values were further decreased when co-cultured with astrocytes. This study documents an increase in the Connexin43 (Cx43) gap junction protein, upon treatment with NTP for both astrocytes and glioblastoma cells. However, treatment with tirapazamine alone ameliorates this effect in glioblastoma cells only and not in astrocytes. The combination therapy of NTP and tirapazamine increased this effect to levels comparable to the original expression levels. The regulation of Cx43 may reduce the metastatic potential of the glioblastoma cells while enabling the combination therapy to show efficacy both between glioblastoma cells and in the context of communication with astrocytes. By this mechanism, the astrocytes may act as conduits to transport ROS between pockets of glioblastoma cells. In summary, the results demonstrate that the novel combination therapy of NTP and tirapazamine is an effective method to selectively target glioblastoma cells with a potential for clinical applications with limited adverse reactions. Citation Format: Matthew Yehl, Andrew Nicolais, Samuel Gerardi, Ahmad Dianat, Olivia Kucharski, Timothy C. Hutcherson, Shoshanna N. Zucker. Combination therapy with nonthermal plasma and tirapazamine selectively targets glioblastoma cells and regulates the gap junction phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1053.

1395-P: The Role of Adipocyte Connexin-43 in Mediating Adipose Tissue Inflammation and Dysfunction in Obesity

Obesity is a leading global health concern and a major risk factor for type-2 diabetes. Thus, identifying mechanisms linking obesity to insulin resistance is of great importance. Chronic low-grade inflammation of the adipose tissue (AT) observed in obesity has been shown as an important pathophysiological link between obesity and the development of systemic insulin resistance, by various mechanisms. Gap junction (GJ) intercellular-communication, primarily composed of connexin- (Cx) 43, has been recently reported to have immunomodulatory roles in various tissues, and increased Cx43 expression and GJ activity have been implicated in the response of various tissues to chronic stress conditions. We have therefore aimed to study the role of Cx43 in shaping the AT adaptive or maladaptive response to these cellular insults of obesity. In a diet-induced obesity mouse model we demonstrated an increase in Cx43 expression in the intact AT, which could be primarily attributed to increased Cx43 expression in adipocytes. In high fat diet-fed adipocyte-specific Cx43 knock-out mice (AdCx43KO) , we observed increased infiltration of macrophages to the AT as compared to wild-type (WT) mice. This was accompanied by morphologic changes in the AT, with increased variability in adipocyte size. While AdCx43KO mice did not significantly differ from WT mice in body weight, adiposity or food intake, the AdCx43KO mice demonstrated reduced whole-body insulin sensitivity assessed by insulin tolerance test. Mechanistically, we observed that adipocytes can interact directly with neighboring adipocytes as well as with macrophages via Cx43-composed gap junctions, an interaction that is likely to be key in attenuation of the tissue inflammatory response. Our results suggest an immunomodulatory and metabolic role for adipocyte-Cx43 in obesity. Disclosure S.Ron: None. I.Ron: None. M.Rathaus: None. R.Livne: None. A.Tirosh: Advisory Panel; Abbott Diagnostics, AstraZeneca, Boehringer Ingelheim International GmbH, Merck & Co., Inc., Novo Nordisk, Sanofi, Consultant; Bayer AG, DreaMed Diabetes, Ltd., Research Support; Medtronic, Speaker's Bureau; Eli Lilly and Company. Funding Israel Science Foundation (922/17)

Connexin 43 Hemichannels Regulate Osteoblast to Osteocyte Differentiation.

Connexin 43 (Cx43) is the predominant connexin subtype expressed in osteocytes. Osteocytes, accounting for 90%–95% of total bone cells, function as orchestrators coordinating balanced activity between bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, two newly developed osteocytic cell lines, OCY454 and IDG-SW3, were used to determine the role of Cx43 gap junctions and hemichannels (HCs) in the regulation of osteoblast to osteocyte differentiation. We found that the Cx43 level was substantially increased during the differentiation of IDG-SW3 cells and is also much higher than that of OCY454 cells. We knocked down Cx43 expression using the lentiviral CRISPR/Cas9 approach and inhibition of Cx43 HCs using Cx43 (E2) antibody in IDG-SW3 cells. Cx43 knockdown (KD) or Cx43 HC inhibition decreased gene expression for osteoblast and osteocyte markers, including alkaline phosphatase, type I collagen, dentin matrix protein 1, sclerostin, and fibroblast growth factor 23, whereas increasing the osteoclastogenesis indicator and the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio at early and late differentiation stages. Moreover, mineralization was remarkably attenuated in differentiated Cx43-deficient IDG-SW3 cells compared to ROSA26 control. The conditioned medium collected from fully differentiated IDG-SW3 cells with Cx43 KD promoted osteoclastogenesis of RAW264.7 osteoclast precursors. Our results demonstrated that Cx43 HCs play critical roles in osteoblast to osteocyte differentiation process and regulate osteoclast differentiation via secreted factors.

Dynamics of Connexin 43 Down Modulation in Human Articular Chondrocytes Stimulated by Tumor Necrosis Factor Alpha.

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (−46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-β1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-β1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.

Protein Kinase C-Mediated Hyperphosphorylation and Lateralization of Connexin 43 Are Involved in Autoimmune Myocarditis-Induced Prolongation of QRS Complex.

Myocarditis is a serious and potentially life-threatening disease, which leads to cardiac dysfunction and sudden cardiac death. An increasing number of evidence suggests that myocarditis is also a malignant complication of coronavirus pneumonia, associated with heart failure and sudden cardiac death. Prolonged QRS complexes that are related to malignant arrhythmias caused by myocarditis significantly increase the risk of sudden cardiac death in patients. However, the molecular mechanisms are not fully known at present. In this study, we identify protein kinase C (PKC) as a new regulator of the QRS complex. In isolated hearts of normal rats, the PKC agonist, phorbol-12-myristate-13-acetate (PMA), induced prolongation of the QRS complex. Mechanistically, hyperphosphorylation and lateralization of connexin 43 (Cx43) by PKC induced depolymerization and internalization of Cx43 gap junction channels and prolongation of the QRS duration. Conversely, administration of the PKC inhibitor, Ro-32-0432, in experimental autoimmune myocarditis (EAM) rats after the most severe inflammation period still significantly rescued the stability of the Cx43 gap junction and alleviated prolongation of the QRS complex. Ro-32-0432 reduced phosphorylation and blocked translocation of Cx43 in EAM rat heart but did not regulate the mRNA expression level of ventricular ion channels and the other regulatory proteins, which indicates that the inhibition of PKC might have no protective effect on ion channels that generate ventricular action potential in EAM rats. These results suggest that the pharmacological inhibition of PKC ameliorates the prolongation of the QRS complex via suppression of Cx43 hyperphosphorylation, lateralization, and depolymerization of Cx43 gap junction channels in EAM rats, which provides a potential therapeutic strategy for myocarditis-induced arrhythmias.

Cx43 hemichannels contribute to astrocyte-mediated toxicity in sporadic and familial ALS

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here, we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In addition, Cx43 expression is up-regulated in human postmortem tissue and cerebrospinal fluid from ALS patients. Using human induced pluripotent stem cell–derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we establish that Cx43 is up-regulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We also demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes using GAP 19, a mimetic peptide blocker, and tonabersat, a clinically tested small molecule, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Extending the in vitro application of tonabersat with chronic administration to SOD1G93A mice results in MN protection with a reduction in reactive astrocytosis and microgliosis. Taking these data together, our studies identify Cx43 hemichannels as conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

Conformational changes and CO2-induced channel gating in connexin26

SummaryConnexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. Opening or closing of the channels is controlled by a variety of stimuli, and dysregulation leads to multiple diseases. An increase in the partial pressure of carbon dioxide (PCO2) has been shown to cause connexin26 (Cx26) gap junctions to close. Here, we use cryoelectron microscopy (cryo-EM) to determine the structure of human Cx26 gap junctions under increasing levels of PCO2. We show a correlation between the level of PCO2 and the size of the aperture of the pore, governed by the N-terminal helices that line the pore. This indicates that CO2 alone is sufficient to cause conformational changes in the protein. Analysis of the conformational states shows that movements at the N terminus are linked to both subunit rotation and flexing of the transmembrane helices.

Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the β3-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

The Amino Terminal Domain and Modulation of Connexin36 Gap Junction Channels by Intracellular Magnesium Ions.

Electrical synapses between neurons in the mammalian CNS are predominantly formed of the connexin36 (Cx36) gap junction (GJ) channel protein. Unique among GJs formed of a number of other members of the Cx gene family, Cx36 GJs possess a high sensitivity to intracellular Mg2+ that can robustly act to modulate the strength of electrical synaptic transmission. Although a putative Mg2+ binding site was previously identified to reside in the aqueous pore in the first extracellular (E1) loop domain, the involvement of the N-terminal (NT) domain in the atypical response of Cx36 GJs to pH was shown to depend on intracellular levels of Mg2+. In this study, we examined the impact of amino acid substitutions in the NT domain on Mg2+ modulation of Cx36 GJs, focusing on positions predicted to line the pore funnel, which constitutes the cytoplasmic entrance of the channel pore. We find that charge substitutions at the 8th, 13th, and 18th positions had pronounced effects on Mg2+ sensitivity, particularly at position 13 at which an A13K substitution completely abolished sensitivity to Mg2+. To assess potential mechanisms of Mg2+ action, we constructed and tested a series of mathematical models that took into account gating of the component hemichannels in a Cx36 GJ channel as well as Mg2+ binding to each hemichannel in open and/or closed states. Simultaneous model fitting of measurements of junctional conductance, gj, and transjunctional Mg2+ fluxes using a fluorescent Mg2+ indicator suggested that the most viable mechanism for Cx36 regulation by Mg2+ entails the binding of Mg2+ to and subsequent stabilization of the closed state in each hemichannel. Reduced permeability to Mg2+ was also evident, particularly for the A13K substitution, but homology modeling of all charge-substituted NT variants showed only a moderate correlation between a reduction in the negative electrostatic potential and a reduction in the permeability to Mg2+ ions. Given the reported role of the E1 domain in Mg2+ binding together with the impact of NT substitutions on gating and the apparent state-dependence of Mg2+ binding, this study suggests that the NT domain can be an integral part of Mg2+ modulation of Cx36 GJs likely through the coupling of conformational changes between NT and E1 domains.

A0439 - Flavonoid nobiletin inhibits IL-1b-induced Cx43 upregulation and gap junction communication in urothelial cells and attenuates Cyclophosphamide induced cystitis in mice

A0439 - Flavonoid nobiletin inhibits IL-1b-induced Cx43 upregulation and gap junction communication in urothelial cells and attenuates Cyclophosphamide induced cystitis in mice

ERGIC2 and ERGIC3 regulate the ER-to-Golgi transport of gap junction proteins in metazoans.

The extremely dynamic life cycle of gap junction connections requires highly efficient intracellular trafficking system especially designed for gap junction proteins, but the underlying mechanisms are largely unknown. Here, we identified that the COPII-associated proteins ERGIC2 (ER-Golgi intermediate compartment) and ERGIC3 are specifically required for the efficient intracellular transport of gap junction proteins in both Caenorhabditis elegans and mice. In the absence of Ergic2 or Ergic3, gap junction proteins accumulate in the ER and Golgi apparatus and the size of endogenous gap junction plaques is reduced. Knocking out the Ergic2 or Ergic3 in mice results in heart enlargement and cardiac malfunction accompanied by reduced number and size of connexin 43 (Cx43) gap junctions. Invertebrates' gap junction protein innexins share no sequence similarity with vertebrates' connexins. However, ERGIC2 and ERGIC3 could bind to gap junction proteins in both worms and mice. Characterization of the highly specialized roles of ERGIC2 and ERGIC3 in metazoans reveals how the early secretory pathway could be adapted to facilitate the efficient transport for gap junction proteins in vivo.

Connexin43 inhibition attenuated dopaminergic neuronal loss in the lipopolysaccharide-induced mice model of Parkinson’s disease

Studies using in vitro Parkinson’s disease (PD) models have found that lipopolysaccharide (LPS) induced reduction of connexin 43 (Cx43) gap junction communication and elevation of hemichannel function, which could cause neurotoxicity directly and indirectly via excessive ATP and glutamate release. However, in vivo study about Cx43 expression and function, as well as the efficacy of Cx43 inhibition for neuronal survival in PD is lacking. This study aimed to unravel the role of Cx43 in PD and understand the underlying mechanisms using an in vivo PD model. Male C57BL/6 mice received intranigral injection of LPS (5 μg) and 43Gap27 (4 μg), a Cx43 inhibitor, simultaneously. Results showed that following LPS treatment, total Cx43 expression decreased by about 60%, but the relative level of phosphorylated Cx43 increased to about double that controls (all p < 0.05). The administration of 43Gap27 significantly attenuated the loss of dopaminergic neurons and restored dopamine and its metabolites levels. Moreover, 43Gap27 treatment inhibited intense microgliosis and astrogliosis in nigrostriatal system induced by LPS and also ameliorated elevated levels of inflammatory mediators. Interestingly, Cx43 inhibition also increased nerve growth factors. In conclusion, Cx43 inhibition was able to prevent LPS-mediated dopaminergic neuronal death, possibly via neuroinflammation reaction reduction and neurotrophic factors elevation. Therefore, Cx43 may be a promising therapeutic target for degenerative neurological disorders such as PD.

Critical Role of Astrocytic Polyamine and GABA Metabolism in Epileptogenesis.

Accumulating evidence indicate that astrocytes are essential players of the excitatory and inhibitory signaling during normal and epileptiform activity via uptake and release of gliotransmitters, ions, and other substances. Polyamines can be regarded as gliotransmitters since they are almost exclusively stored in astrocytes and can be released by various mechanisms. The polyamine putrescine (PUT) is utilized to synthesize GABA, which can also be released from astrocytes and provide tonic inhibition on neurons. The polyamine spermine (SPM), synthesized form PUT through spermidine (SPD), is known to unblock astrocytic Cx43 gap junction channels and therefore facilitate astrocytic synchronization. In addition, SPM released from astrocytes may also modulate neuronal NMDA, AMPA, and kainate receptors. As a consequence, astrocytic polyamines possess the capability to significantly modulate epileptiform activity. In this study, we investigated different steps in polyamine metabolism and coupled GABA release to assess their potential to control seizure generation and maintenance in two different epilepsy models: the low-[Mg2+] model of temporal lobe epilepsy in vitro and in the WAG/Rij rat model of absence epilepsy in vivo. We show that SPM is a gliotransmitter that is released from astrocytes and significantly contributes to network excitation. Importantly, we found that inhibition of SPD synthesis completely prevented seizure generation in WAG/Rij rats. We hypothesize that this antiepileptic effect is attributed to the subsequent enhancement of PUT to GABA conversion in astrocytes, leading to GABA release through GAT-2/3 transporters. This interpretation is supported by the observation that antiepileptic potential of the Food and Drug Administration (FDA)-approved drug levetiracetam can be diminished by specifically blocking astrocytic GAT-2/3 with SNAP-5114, suggesting that levetiracetam exerts its effect by increasing surface expression of GAT-2/3. Our findings conclusively suggest that the major pathway through which astrocytic polyamines contribute to epileptiform activity is the production of GABA. Modulation of astrocytic polyamine levels, therefore, may serve for a more effective antiepileptic drug development in the future.