Functional Gap Junctions Research Articles

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva.

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

A Broad-Spectrum Chemokine Inhibitor Blocks Inflammation-Induced Myometrial Myocyte-Macrophage Crosstalk and Myometrial Contraction.

Prophylactic administration of the broad-spectrum chemokine inhibitor (BSCI) FX125L has been shown to suppress uterine contraction, prevent preterm birth (PTB) induced by Group B Streptococcus in nonhuman primates, and inhibit uterine cytokine/chemokine expression in a murine model of bacterial endotoxin (LPS)-induced PTB. This study aimed to determine the mechanism(s) of BSCI action on human myometrial smooth muscle cells. We hypothesized that BSCI prevents infection-induced contraction of uterine myocytes by inhibiting the secretion of pro-inflammatory cytokines, the expression of contraction-associated proteins and disruption of myocyte interaction with tissue macrophages. Myometrial biopsies and peripheral blood were collected from women at term (not in labour) undergoing an elective caesarean section. Myocytes were isolated and treated with LPS with/out BSCI; conditioned media was collected; cytokine secretion was analyzed by ELISA; and protein expression was detected by immunoblotting and immunocytochemistry. Functional gap junction formation was assessed by parachute assay. Collagen lattices were used to examine myocyte contraction with/out blood-derived macrophages and BSCI. We found that BSCI inhibited (1) LPS-induced activation of transcription factor NF-kB; (2) secretion of chemokines (MCP-1/CCL2 and IL-8/CXCL8); (3) Connexin43-mediated intercellular connectivity, thereby preventing myocyte–macrophage crosstalk; and (4) myocyte contraction. BSCI represents novel therapeutics for prevention of inflammation-induced PTB in women.

Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines.

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

Inhibition of gap junction communication between cells can induce apoptosis of corpus cavernosum smooth muscle in guinea pigs.

In this study, we aimed to investigate the effect of gap junction (GJ) on apoptosis of smooth muscle. Forty adult male guinea pigs were randomly divided into four groups with 10 guinea pigs in each group. Adeno-associated virus (AAV) and Gap27 were injected at the root of the corpus cavernosum. Two weeks later, the corpus cavernosum tissue was taken to be tested. The expression of Cx43 and α-SMC protein was detected by immunofluorescence and Western blotting. The content of corpus cavernosum smooth muscle was detected by Masson trichrome staining. Apoptosis was detected by TUNEL staining and Western blotting. The results showed that Gap27 did not affect Cx43 but decreased the expression of smooth muscle. The results of TUNEL staining and detection of apoptosis-related proteins showed that apoptosis was induced by Gap27. In addition, we found that corpus cavernosum injection of AAV could induce obvious apoptosis. In this study, we examined the effect of inhibition of gap junction on smooth muscle, and suggested that the decrease of gap junction function may be a potential mechanism of smooth muscle apoptosis.

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals.

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

Core-shell nanofibre scaffold mediated co-delivery of connexin-43 gene and histone deacetylase inhibitor for anticancer therapy

Connexin 43 (Cx43), a gap junction protein, also acts as a tumor suppressor, which reduces tumorigenicity. Cx43 expression sensitizes cancer cells towards anticancer drugs mainly due to the formation of functional gap junctions. 4-phenyl butyrate (4-PB), a histone deacetylase inhibitor (HDACi), has antitumor potential that is also reported to increase Cx43 expression and gap junction communication. In the present work, we have developed a core-shell nanofibrous scaffold loaded with the Cx43 gene and 4-PB. The nanofibrous scaffold has been characterized by spectroscopic and electron microscopic techniques. The nanofibrous scaffold mediated transfection of the Cx43 gene into Cx43 deficient breast cancer cells (MCF-7), followed by controlled and sustained release of 4-PB, exhibited anti-cell proliferative activity. The anticancer effects have been ascertained by cell staining, cell viability and flow cytometry-based assays. Thus, the novel core-shell nanofiber scaffold has shown enormous therapeutic translation ability via Cx-based enhancement of anticancer potential of HDACi.

Glioblastoma-Astrocyte Connexin 43 Gap Junctions Promote Tumor Invasion.

Glioblastoma multiforme (GBM), classified as World Health Organization grade IV astrocytoma, is the deadliest adult cancer of the central nervous system. An important contributing factor to poor survival rates in GBM is extensive invasion, which decreases the efficacy of resection and subsequent adjuvant therapies. These treatments could be markedly improved with increased resolution of the genetic and molecular initiators and effectors of invasion. Connexin 43 (Cx43) is the principal astrocytic gap junction (GJ) protein. Despite the heterogeneity of GBM, a subpopulation of cells in almost all GBM tumors express Cx43. Functional GJs between GBM cells and astrocytes at the tumor edge are of critical interest for understanding invasion. In this study, we find that both in vitro and in ex vivo slice cultures, GBM is substantially less invasive when placed in a Cx43-deficient astrocyte environment. Furthermore, when Cx43 is deleted in GBM, the invasive phenotype is recovered. These data strongly suggest that there are opposing roles for Cx43 in GBM migration. We find that Cx43 is localized to the tumor edge in our ex vivo model, suggesting that GBM-astrocyte GJ communication at the tumor border is a driving force for invasion. Finally, we find that by a Cx43-dependent mechanism, but likely not direct channel-mediated diffusion, miRNAs associated with cell-matrix adhesion are transferred from GBM to astrocytes and miR-19b promotes invasion, revealing a role for post-transcriptional manipulation of astrocytes in fostering an invasion-permissive peritumoral niche. IMPLICATIONS: Cx43-mediated communication, specifically miRNA transfer, profoundly impacts glioblastoma invasion and may enable further therapeutic insight.

A gap-junction-mediated, calcium-signaling network controls blood progenitor fate decisions in hematopoiesis

Stem cell homeostasis requires coordinated fate decisions among stem cells that are often widely distributed within a tissue at varying distances from their stem cell niche. This requires a mechanism to ensure robust fate decisions within a population of stem cells. Here, we show that, in the Drosophila hematopoietic organ, the lymph gland (LG), gap junctions form a network that coordinates fate decisions between blood progenitors. Using live imaging of calcium signaling in intact LGs, we find that blood progenitors are connected through a signaling network. Blocking gap junction function disrupts this network, alters the pattern of encoded calcium signals, and leads to loss of progenitors and precocious blood cell differentiation. Ectopic and uniform activation of the calcium-signaling mediator CaMKII restores progenitor homeostasis when gap junctions are disrupted. Overall, these data show that gap junctions equilibrate cell signals between blood progenitors to coordinate fate decisions and maintain hematopoietic homeostasis.

Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell-Cell Communication?

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

IPSC-Derived Trabecular Meshwork Cells Stimulate Endogenous TM Cell Division Through Gap Junction in a Mouse Model of Glaucoma

PurposeDecreased trabecular meshwork (TM) cellularity has been implicated as a major reason for TM dysfunction and aqueous humor (AH) outflow abnormalities in primary open angle glaucoma. We previously found that transplantation of induced pluripotent stem cell (iPSC)-derived TM cells can restore TM function and stimulate endogenous TM cell division. The goal of the present study is to investigate whether signaling via gap junctions is involved in this process.MethodsDifferentiated iPSCs were characterized morphologically, transcriptionally, and immunohistochemically. After purification, iPSC-TM were co-cultured with mouse TM (MTM) cells to mimic the transplantation procedure. Through the pharmacological antagonists and short hairpin RNA (shRNA) technique, the gap junction function in iPSC-based therapy was determined.ResultsIn the co-culture system, iPSC-TM increase MTM cell division as well as transfer of Ca2+ to MTM. This effect was blocked by treatment with the gap junction inhibitors carbenoxolone (CBX) or flufenamic acid (FFA). The shRNA mediated knock down of connexin 43 (Cx43) expression in iPSC-TM also results in decreased Ca2+ transfer and lower MTM proliferation rates. In vivo, Cx43 downregulation in transplanted iPSC-TM weakened their regenerative role in an Ad5.myocilinY437H mouse model of glaucoma. Mice receiving these cells exhibited lower TM cellularity and higher intraocular pressure (IOP) than those receiving unmodified iPSC-TM.ConclusionsOur findings reveal a crucial role of gap junction, especially Cx43, in iPSC-based TM regeneration, and provides insights to enhance the regenerative effect of iPSCs in glaucoma therapy.

Effect of berbamine on invasion and metastasis of human liver cancer SMMC-7721 cells and its possible mechanism.

Berbamine is a bisbenzylisoquinoline alkaloid extracted from Berberis poiretii of Berberis of Berberidaceae. It has been reported that it can significantly inhibit the proliferation of a variety of malignant tumor cells, including liver cancer. However, the effect of berbamine on the invasion and metastasis of liver cancer has not been reported. The present study demonstrated that berbamine inhibited the migration and invasion of SMMC-7721 cells in a concentration-dependent manner and obviously increased the gap junction function and the expression of Cx32 in SMMC-7721 cells compared with control group. However, after silencing Cx32, berbamine had no significant effect on cell invasion and metastasis. Before silencing Cx32, the expression of PI3K and P-AKT were decreased after berbamine treated on SMMC-7721 cells for 24 h. After silencing Cx32, the expression of PI3K and P-AKT were increased in SMMC-7721 cells. The expression of PI3K and P-AKT had no significant effect after berbamine treated on SMMC-7721 cells for 24 h with silencing Cx32. In conclusion, the results of the present study suggest that berbamine could inhibit the SMMC-7721 cell migration and invasion, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway by enhancing the expression of Cx32.

Korean red ginseng alleviate depressive disorder by improving astrocyte gap junction function

Korean red ginseng (KRG), a processed product of Panax ginseng C. A. Mey, show significant anti-depressive effect in clinic. However, its mechanism is still unclear. Gap junction intercellular communication (GJIC) dysfunction is a potential pathogenesis of depression. Therefore, this study's objective is to investigate whether the antidepressant effect of KRG is related to GJIC. Rat were restraint 8h every day for 28 consecutive days to prepare depression models, and meanwhile, rats were intragastrically administrated with normal saline, KRG solutions (25, 50 or 100mg/kg) or fluoxetine (10mg/kg) 1h before stress. The behavioral performance was determined by forced swimming test, sucrose preference test and open field test. GJIC was determined by the Lucifer yellow (LY) diffusion distance in prelimb cortex (PLC). In addition, the level of Cx43, one of executors of GJIC, was tested by Western blot. To find out the protective effect of KRG against GJIC dysfunction directly, rats were intracranially injected with carbenoxolone (CBX, blocker of GJIC), and meanwhile normal saline, KRG (100mg/kg) or fluoxetine (10mg/kg) was administered daily. The behavioral performance of these rats was detected, and the LY localization injection PLC area was used to detect the gap junction function. Chronic resistant stress (CRS) induced depressive symptoms, as manifested by prolonged immobility time in forced swimming test and decreased sucrose consumption ratio. Administration of KRG alleviated these depressive symptoms significantly. GJIC determination showed that KRG improved the LY diffusion and increased Cx43 level in prefrontal cortex (PFC) significantly, indicated that GJIC dysfunction was alleviated by the treatment of KRG. However, the astrocytes number was also increased by the treatment of KRG, which maybe alleviate depression-like symptoms by increasing the number of astrocytes rather than improving GJIC. Injection of CBX produced depressive symptoms and GJIC dysfunction, as manifested by decreased sucrose consumption ratio and prolonged immobility time in forced swimming test, but no astrocytes number changes, KRG also reversed depressive symptoms and GJIC dysfunction, suggested that the improvement of depressive-like symptoms was improved by GJIC. KRG alleviate depressive disorder by improving astrocytic gap junction function.

Abstract 2885: Connexin 43-dependent miRNA transfer drives perivascular glioma invasion through dysregulation of astrocytes

Abstract Background: Glioblastoma multiforme (GBM) invasion occurs along vasculature, and intercellular communication between glioma cells and the astrocyte endfoot may manipulate astrocytes to repress cell-matrix adhesion through transfer of miRNAs. Intercellular communication, specifically through Connexin 43 (Cx43) gap junction (GJ) channels, has been confoundingly demonstrated to both limit and facilitate GBM invasion. In solid tumors, Cx43 between tumor cells is correlated with a low invasion and the GJs have long been postulated to be required for growth control. By contrast, functional Cx43 channels between GBM and non-cancerous cells is correlated with high invasiveness, and GJs have been proposed to provide the pathway through which GBM drives surrounding astrocytes into a tumor-permissive state. How GJs participate in the transfer of genetic regulatory molecules from GBM to astrocytes is incompletely understood and might involve direct diffusion through Cx43 channels, GJ-facilitated exosomal transfer, and biased GJ endocytosis. In this study we examine astrocyte miRNA expression after contact with GBM and used an ex vivo assay to assess Cx43-dependent invasion. Methods: Immortalized wild-type (WT) and connexin 43 (Cx43) knockout (KO) murine astrocytes were co-cultured for 24 hours with U87 GBM cells expressing cytosolic GFP. Cells were separated by FACS and collected for miRNAseq. For ex vivo invasion model, 350µm whole brain coronal slices (from WT and GFAP-Cre Cx43-/- C57BL/6J mice) were obtained via vibratome in high-sucrose ACSF and cultured for >12 hours in 20% serum recovery medium. At that time, 250-350µm U87 spheroids (4-8 per slice) expressing cytosolic mCherry (either WT or CRISPR-generated Cx43 KO U87 cell lines) were injected into the cerebral cortex. Slices were cultured for up to 72 hours, fixed, and cleared by SeeDB protocol. Invasion was imaged by confocal microscopy at 10x. Extent of perivascular invasion by the injected tumor spheroids was quantified using Sholl analysis to compare number and lengths of branches. Results: miRNASeq identified a cohort of >10 miRNAs that were upregulated in WT astrocytes, but not KO astrocytes, co-cultured with GBM. Some of these miRNAs are known to have elevated expression in GBM and have predicted cell adhesion protein targets. GBM pervascular invasion was substantially lower for WT-U87 spheroids in GFAP-Cre Cx43-/- slice when compared to WT-U87 in WT slice or Cx43 KO U87 in Cx43-/- slice. Conclusions: Specific miRNAs are transferred via Cx43-mediated mechanisms from GBM to astrocytes and may play a role in astrocyte-matrix adhesion at the endfoot, creating an opening for GBM invasion. Initial slice culture data indicate that functional GBM-GBM GJs inhibit peritumoral outgrowth, whereas astrocyte-GBM GJs are critical for invasion, presumably facilitating remodeling of host tissue by the miRNAs to accommodate invading GBM. Citation Format: Sean McCutcheon, David C. Spray. Connexin 43-dependent miRNA transfer drives perivascular glioma invasion through dysregulation of astrocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2885.

A Nuclear and Cytoplasmic Characterization of Bovine Oocytes Reveals That Cysteamine Partially Rescues the Embryo Development in a Model of Low Ovarian Reserve.

Decreased oocyte quality is a major determinant of age-associated fertility decline. Similarly, individuals affected by early ovarian aging carry low-quality oocytes. Using an established bovine model of early ovarian aging, we investigated key features of 'quality' oocyte maturation, associated with the onset of egg aneuploidy and reproductive aging, such as histone modifications, mitochondria distribution and activity, reduced glutathione (GSH) content, and gap junction functionality. Bovine ovaries were classified according to the antral follicle count (AFC), and the retrieved oocytes were processed immediately or matured in vitro. We observed alterations in several cellular processes, suggesting a multifactorial etiology of the reduced oocyte quality. Furthermore, we performed a rescue experiment for one of the parameters considered. By adding cysteamine to the maturation medium, we experimentally increased the free radical scavenger ability of the 'low competence' oocytes and obtained a higher embryo development. Our findings show that adopting culture conditions that counteract the free radicals has a positive impact on the quality of 'compromised' oocytes. Specifically, cysteamine treatment seems to be a promising option for treating aging-related deficiencies in embryo development.

Connexin 43 contributes to phenotypic robustness of the mouse skull.

We compared skull shape and variation among genetically modified mice that exhibit different levels of connexin43 (Cx43) channel function, to determine whether Cx43 contributes to craniofacial phenotypic robustness. Specifically, we used two heterozygous mutant mouse models (G60S/+ and I130T/+) that, when compared to their wildtype counterparts, have an ~80% and ~50% reduction in Cx43 function, respectively. Both mutant strains showed significant differences in skull shape compared to wildtype littermates and while these differences were more severe in the G60S/+ mouse, shape differences were localized to similar regions of the skull in both mutants. However, increased skull shape variation was observed in G60S/+ mutants only. Additionally, covariation of skull structures was disrupted in the G60S/+ mutants only, indicating that while a 50% reduction in Cx43 function is sufficient to cause a shift in mean skull shape, the threshold for Cx43 function for disrupting craniofacial phenotypic robustness is lower. Collectively, our results indicate Cx43 can contribute to phenotypic robustness of the skull through a nonlinear relationship between Cx43 gap junctional function and phenotypic outcomes.

Assessment of cGMP level in medium during in vitro growth period of murine preantral follicles with and without supplementation of C-type natriuretic peptide.

To enhance the developmental competency of murine ovarian follicles cultured in vitro, C-type natriuretic peptide (CNP) was supplemented in the culture system. Although the mechanism is not fully elucidated, it was reported that the effect of CNP supplementation was mediated by increased cyclic guanosine monophosphate (cGMP). In the present study, cGMP levels in media for murine preantral follicle culture were compared both between a control group without CNP supplementation and an experimental group with CNP supplementation and between days in each group. In addition, follicle growth patterns and oocyte maturity were assessed and compared between the two groups. Results demonstrated that along with in vitro culture, cGMP levels increased (P < 0.05) both in the control group and the experimental group, whereas cGMP levels were not significantly different between the two groups on the same day of in vitro culture (P > 0.05). The oocyte's maturity was superior in the experimental group compared with the control group (P < 0.05). As ovarian follicles grew three-dimensionally in the experimental group but were flattened in the control group, CNP might improve oocyte maturity through maintaining the three-dimensional architecture of the ovarian follicle because of increased transzonal projections (TZP) and functional gap junctions between oocyte and surrounding granulosa cells.

Cysteine residues in the C-terminal tail of connexin32 regulate its trafficking

Gap junctions (GJs) are formed by the assembly of constituent transmembrane proteins called connexins (Cxs). Aberrations in this assembly of Cxs are observed in several genetic diseases as well as in cancers. Hence it becomes imperative to understand the molecular mechanisms underlying such assembly defect. The polarized cells in the epithelia express Connexin32 (Cx32). The C-terminal tail (CT) of Cx32 orchestrates several aspects of GJ dynamics, function and growth. The study here was aimed at determining if post-translational modifications, specifically, palmitoylation of cysteine residues, present in the CT of Cx32, has any effect on GJ assembly. The CT of Cx32 was found to harbor three cysteine residues, which are likely to be modified by palmitoylation. The study here has revealed for the first time that Cx32 is palmitoylated at cysteine 217 (C217) in cell line derived from prostate tumors. However, it was found that mutating C217 to alanine affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, it was discovered that mutating cysteine 280 and 283, only in combination, blocked the trafficking of Cx32 from the trans-Golgi network to the cell surface. The mutants showed reduced stability due to enhanced lysosomal degradation. Overall, the findings reveal the importance of the two C-terminal cysteine residues of Cx32 in regulating its trafficking and stability and hence its ability to assemble into GJs.

Connexin 46 and connexin 50 gap junction channel properties are shaped by structural and dynamic features of their N-terminal domains.

Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies by cryo-electron microscopy have produced high-resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure-function comparison. Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform-specific differences in Cx46 and Cx50 intercellular channel function. We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites. The results of this study establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease-causing variants. Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long-range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino-terminal (NT) domain as a major contributor for isoform-specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46-50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free-energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage-dependent gating (Vj gating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra- and inter-subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract-linked connexin variants.

Extracellular vesicles regulate gap junction-mediated intercellular communication and HIV-1 infection of human neural progenitor cells

Human immunodeficiency virus-1 (HIV-1) has been shown to cross the blood-brain barrier and cause HIV-associated neurocognitive disorders (HAND) through a process that may involve direct or indirect interactions with the central nervous system (CNS) cells and alterations of amyloid β (Aβ) homeostasis. The present study focused on the mechanisms of HIV-1 infecting human neural progenitor cells (hNPCs) and affecting NPC intercellular communications with human brain endothelial cells (HBMEC). Despite the lack of the CD4 receptor, hNPCs were effectively infected by HIV-1 via a mechanism involving the chemokine receptors, CXCR4 and CCR5. HIV-1 infection increased expression of connexin-43 (Cx43), phosphorylated Cx43 (pCx43), and pannexin 2 (Panx2) protein levels in hNPCs, suggesting alterations in gap-junction (GJ) and pannexin channel communication. Indeed, a functional GJ assay indicated an increase in communication between HIV-infected hNPCs and non-infected HBMEC. We next analyzed the impact of HBMEC-derived extracellular vesicles (EVs) and EVs carrying Aβ (EV-Aβ) on the expression of Cx43, pCx43, and Panx2 in HIV-1 infected and non-infected hNPCs. Exposure to EV-Aβ resulted in significant reduction of Cx43 and pCx43 protein expression in non-infected hNPCs when compared to EV controls. Interestingly, EV-Aβ treatment significantly increased levels of Cx43, pCx43, and Panx2 in HIV-1-infected hNPCs when compared to non-infected controls. These results were confirmed in a GJ functional assay and an ATP release assay, which is an indicator of connexin hemichannel and/or pannexin channel functions. Overall, the current study demonstrates the importance of hNPCs in HIV-1 infection and indicates that intercellular communications between infected hNPCs and HBMEC can be effectively modulated by EVs carrying Aβ as their cargo.

Electrical synaptic transmission requires a postsynaptic scaffolding protein.

Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses.