Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC.
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ABSTRACT:
SUBMITTER: Bao X
PROVIDER: S-EPMC1817834 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to "chemical ischemia" by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18alpha-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La(3+) also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.
| S-EPMC117588 | biostudies-literature
Project description:Gap junction channels in cumulus-oocyte complexes (COCs) enable the transmission and communication of small molecular signals between adjacent cells, such as cAMP. However, the regulation of gap junction function (GJF) by cAMP and the underlying mechanisms involved are not fully clarified. This study investigated the effect of cAMP on connexin 43 (CX43) expression and GJF in ovine COCs using immunofluorescence, quantitative real-time PCR (qRT-PCR), western blotting, and GJF detection. The CX43 was only found in the cumulus cells (CCs) side of ovine COC. The intra-oocyte cAMP showed a significant increase at 30 min, while the intra-CC cAMP exhibited two peaks at 10 min and 1 h during in vitro maturation (IVM). Phosphorylated CX43 protein exhibited an immediate increase at 10 min, and CX43 protein displayed two peaks at 10 min and 1 h during IVM. The duration of pre-IVM exposure to forskolin and IBMX significantly enhanced phosphorylated and total CX43, as well as Gja1 and Creb genes, for 10 min; these effects were counteracted by Rp-cAMP. Both pre-IVM with forskolin and IBMX for 1 h and the GJF and CX43/p-CX43 ratio were elevated. The closure of gap junction channels caused by phosphorylated CX43 to prevent cAMP outflow from oocytes in early IVM of COC. Cyclic AMP upregulated phosphorylated and total CX43 via genomic and non-genomic pathways, but its functional regulation was dependent on the balance of the two proteins. This study provides a new insight into the regulatory mechanism between cAMP and GJF, which would improve IVM in animal and clinical research.
| S-EPMC10620458 | biostudies-literature
Project description:Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4',6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.
| S-EPMC3118918 | biostudies-literature
Project description:Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.
| S-EPMC2755956 | biostudies-literature
Project description:Connexin 43 (Cx43) nonjunctional or "unapposed" hemichannels can open under physiological or pathological conditions. We characterize hemichannels comprised of Cx43 or Cx43-EGFP (Cx43 with enhanced GFP fused to the C terminus) expressed in HeLa cells. Channel opening was induced at potentials greater than +60 mV. Open probability appeared to be very low. No comparable opening was detected in the parental, nontransfected HeLa cells. Conductance of fully open single hemichannels was approximately 220 pS, which is approximately double that of Cx43 cell-cell channels. Cx43 hemichannels exhibited two types of gating: fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS and slow transitions (>5 ms) between either open state and the fully closed state. Cx43-EGFP hemichannels exhibited only slow transitions (>5 ms) between closed and fully open states. These properties resemble those of the corresponding Cx43 and Cx43-EGFP cell-cell channels. Cx43 with EGFP on the N terminus (EGFP-Cx43) inserted into the surface and formed plaques but did not form hemichannels or cell-cell channels. Hemichannel blockers, 18beta-glycyrrhetinic acid or La3+, blocked depolarization-induced currents. Uptake of ethidium bromide (i) was faster in Cx43 and Cx43-EGFP than parental and EGFP-Cx43 cells, (ii) was directly correlated with Cx43-EGFP expression, (iii) was reduced by hemichannel blockers, and (iv) occurred at the same low rate in EGFP-Cx43 and parental cells. Although hemichannel opening was not detected electrophysiologically at the resting potential, infrequent or brief opening could account for ethidium bromide uptake. Opening of Cx43 hemichannels may mediate normal signaling or be deleterious.
| S-EPMC208767 | biostudies-literature
Project description:The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in various tumors promotes cell proliferation and invasiveness. The intracellular cellular pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by characterizing the the interactome of recombinant ACKR3 expressed in HEK293T cells. Among the ACKR3-interacting proteins identified, we focused on the gap junction protein Connexin 43 (Cx43).
2020-07-07 | PXD016978 | Pride
Project description:The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes β-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.
| S-EPMC7519114 | biostudies-literature
Project description:Estrogen deficiency in postmenopausal women is a major cause of bone loss, resulting in osteopenia, osteoporosis, and a high risk for bone fracture. Connexin 43 (Cx43) hemichannels (HCs) in osteocytes play an important role in osteocyte viability, bone formation, and remodeling. We showed here that estrogen deficiency reduced Cx43 expression and HC function. To determine if functional HCs protect osteocytes and bone loss during estrogen deficiency, we adopted an ovariectomy model in wild-type (WT) and two transgenic Cx43 mice: R76W (dominant-negative mutant inhibiting only gap junction channels) and Cx43 Δ130-136 (dominant-negative mutant compromising both gap junction channels and HCs). The bone mineral density (BMD), bone structure, and histomorphometric changes of cortical and trabecular bones after ovariectomy were investigated. Our results showed that the Δ130-136 transgenic cohort had greatly decreased vertebral trabecular bone mass compared to WT and R76W mice, associated with a significant increase in the number of apoptotic osteocyte and empty lacunae. Moreover, osteoclast surfaces in trabecular and cortical bones were increased after ovariectomy in the R76W and WT mice, respectively, but not in ∆130-136 mice. These data demonstrate that impairment of Cx43 HCs in osteocytes accelerates vertebral trabecular bone loss and increase in osteocyte apoptosis, and further suggest that Cx43 HCs in osteocytes protect trabecular bone against catabolic effects due to estrogen deficiency.
| S-EPMC6476886 | biostudies-literature
Project description:The metabolic syndrome, which comprises obesity and diabetes, is a major public health problem and the awareness of energy homeostasis control remains an important worldwide issue. The energy balance is finely regulated by the central nervous system (CNS), notably through neuronal networks, located in the hypothalamus and the dorsal vagal complex (DVC), which integrate nutritional, humoral and nervous information from the periphery. The glial cells' contribution to these processes emerged few year ago. However, its underlying mechanism remains unclear. Glial connexin 43 hemichannels (Cx43 HCs) enable direct exchange with the extracellular space and can regulate neuronal network activity. In the present study, we sought to determine the possible involvement of glial Cx43 HCs in energy balance regulation. We here show that Cx43 is strongly expressed in the hypothalamus and DVC and is associated with glial cells. Remarkably, we observed a close apposition of Cx43 with synaptic elements in both the hypothalamus and DVC. Moreover, the expression of hypothalamic Cx43 mRNA and protein is modulated in response to fasting and diet-induced obesity. Functionally, we found that Cx43 HCs are largely open in the arcuate nucleus (ARC) from acute mice hypothalamic slices under basal condition, and significantly inhibited by TAT-GAP19, a mimetic peptide that specifically blocks Cx43 HCs activity. Moreover, intracerebroventricular (i.c.v.) TAT-GAP19 injection strongly decreased food intake, without further alteration of glycaemia, energy expenditures or locomotor activity. Using the immediate early gene c-Fos expression, we found that i.c.v. TAT-GAP19 injection induced neuronal activation in hypothalamic and brainstem nuclei dedicated to food intake regulation. Altogether, these results suggest a tonic delivery of orexigenic molecules associated with glial Cx43 HCs activity and a possible modulation of this tonus during fasting and obesity.
| S-EPMC7693394 | biostudies-literature
Project description:The gap junction protein Connexin 43 (Cx43) contributes to cell fate decisions that determine the location of fin ray joints during regeneration. Here, we provide insights into how Cx43, expressed medially, influences changes in gene expression in lateral skeletal precursor cells. Using the Gap27 peptide inhibitor specific to Cx43, we show that Cx43-gap junctional intercellular communication (GJIC) influences Cx43-dependent skeletal phenotypes, including segment length. We also demonstrate that Cx43-GJIC influences the expression of the Smp/β-catenin pathway in the lateral skeletal precursor cells, and does not influence the Sema3d pathway. Moreover, we show that the cx43lh10 allele, which has increased Cx43 protein levels, exhibits increased regenerate length and segment length. These phenotypes are rescued by Gap27, suggesting that increased Cx43 is responsible for the observed Cx43 phenotypes. Finally, our findings suggest that inhibition of Cx43 hemichannel activity does not influence Cx43-dependent skeletal phenotypes. These data provide evidence that Cx43-GJIC is responsible for regulating cell fate decisions associated with appropriate joint formation in the regenerating fin.
| S-EPMC7338267 | biostudies-literature