Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.
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To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways. (+info)
Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels.
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Connexin channels are gated by transjunctional voltage (Vj) or CO2 via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT1) of connexin32 (Cx32) were shown to determine CO2 sensitivity, and a gating mechanism involving CL-CT1 association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT1 interactions, display atypical Vj and CO2 sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH2-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT1 arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to Vj polarity, the existence of a "slow gate" distinct from the conventional Vj gate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO2 sensitive than homotypic Cx32 channels, and CO2-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting Vj sensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed. (+info)
Molecular mechanism underlying a Cx50-linked congenital cataract.
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Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526-532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of approximately 5 microS 4-7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels when coinjected with wild-type connexin 50 cRNA. Cells injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA exhibited gap junctional coupling of approximately 8% and 39% of wild-type coupling, respectively. Based on these findings, we conclude that only one P88S mutant subunit is necessary per gap junctional channel to abolish channel function. (+info)
ATP is a mediator of the fast inhibitory junction potential in human jejunal circular smooth muscle.
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The neurotransmitter(s) that generates the fast component of the inhibitory junction potential (IJP-F) in human jejunal circular smooth muscle is not known. The aim of this study was to determine the role of ATP and purinergic receptors in the generation of the IJP-F in human jejunal circular smooth muscle strips. The P2-receptor antagonist suramin (100 microM) reduced the IJP-F by 28%. Apamin (1 microM) reduced the IJP-F by 25%. Desensitization of muscle strips with the putative P2x-receptor agonist alpha, beta-methylene ATP (alpha,beta-MeATP, 100 microM) decreased the IJP-F by 44%, and desensitization with the putative P2y-receptor agonist adenosine 5'-O-2-thiodiphosphate (ADPbetaS) completely abolished the IJP-F. Desensitization with the putative P2y-receptor agonist 2-methylthioATP had no effect on the IJP-F. Exogenous ATP evoked a hyperpolarization with a time course that matched the IJP-F. The ATP-evoked hyperpolarization was reduced by apamin and suramin, reduced by desensitization with alpha,beta-MeATP (69% decrease), and abolished by desensitization with ADPbetaS. These data suggest that the IJP-F in human jejunal circular smooth muscle is mediated in part by ATP through an ADPbetaS-sensitive P2 receptor. (+info)
Further evidence for the selective disruption of intercellular communication by heptanol.
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The lack of selective gap junctional uncoupling agents has hampered evaluation of the contribution of intercellular communication to pharmacomechanical coupling and vascular contractility. Thus we further explored the utility and selectivity of heptanol as a gap junctional uncoupling agent in isolated rat aortic rings. Fifty-two aortic rings were obtained from 15 rats and were precontracted to approximately 75% of maximum with phenylephrine (PE). When contraction achieved steady state (approximately 5 min), a single concentration of heptanol (200 microM) was added to each aortic ring at 1- to 3-min intervals for up to 42 min post-PE addition. At early time points (5-10 min after PE), heptanol elicited an approximately 50% loss of tension (i.e., relaxation). At subsequent time points post-PE, a gradual and time-dependent decrease in the magnitude of the heptanol-induced relaxation was observed until, after approximately 40 min, addition of heptanol was associated with little, if any, detectable relaxation. Linear regression analysis of the magnitude of the heptanol-induced relaxation vs. the square root of the elapsed time interval (from addition of PE) revealed a highly significant negative correlation (P < 0.001, R = 0.81). Studies conducted on KCl-precontracted aortic rings revealed no detectable heptanol-induced relaxation after development of the steady-state KCl-induced contraction. These data extend our previous observations to further document the potential utility of heptanol as a "relatively selective" uncoupling agent. (+info)
Inhibition of gap junction communication in alveolar epithelial cells by 18alpha-glycyrrhetinic acid.
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Cultured alveolar epithelial cells exhibit gap junction intercellular communication (GJIC) and express regulated levels of connexin (Cx) 43 mRNA and protein. Newly synthesized radiolabeled Cx43 protein equilibrates with phosphorylated Cx43 isoforms; these species assemble to form both connexons and functional gap junction plaques. The saponin 18alpha-glycyrrhetinic acid (GA) rapidly and reversibly blocks GJIC at low concentrations (5 microM). Extended exposure to 18alpha-GA at higher concentrations causes inhibition of GJIC and time- and dose-dependent reductions in both Cx43 protein and mRNA expression. The latter toxic effects are paralleled by disassembly of gap junction plaques and are reversed less readily than acute effects on GJIC. These observations demonstrate 18alpha-GA-sensitive regulation of intercellular communication in epithelial cells from the mammalian lung and suggest a role for Cx43 expression and phosphorylation in acute and chronic regulation of GJIC between alveolar epithelial cells. (+info)
Multiple connexins form gap junction channels in rat basilar artery smooth muscle cells.
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Three connexins, Cx43, Cx40, and Cx37, have been found by protein or mRNA analysis to be prominent in mammalian blood vessels, but electrophysiological characterization of gap junction channels in freshly isolated vascular smooth muscle cells (SMCs) has not previously been reported. We used a dual-perforated patch-clamp technique to study gap junction conductances in SMC pairs from rat basilar arteries. Macroscopic junctional conductance (Gj) measured in 98 cell pairs with either Cs+ or K+ ranged between 0.68 and 24.8 nS. In weakly coupled cells (Gj<5 nS), single-channel currents were readily resolved without pharmacological uncoupling agents, allowing identification of 4 major unitary conductances. Two of these conductances, 80 to 120 pS and 150 to 200 pS, corresponded to the major conductance states for homotypic channels formed from Cx43 or Cx40, which we confirmed were present in smooth muscle by immunofluorescence analysis. Two other conductances, 220 to 280 pS and >300 pS, were identified that have not been previously reported in vascular SMCs. Macroscopic recordings revealed currents that deactivated incompletely over a broad range of transjunctional potentials. In about half of the pairs, we identified macroscopic as well as single-channel currents that exhibited marked voltage asymmetry, consistent with nonhomotypic, ie, either heterotypic or heteromeric channels. Our data indicate that basilar artery SMCs are coupled in vivo in a richly complex manner, involving Cx43, Cx40, and other large-conductance channels, and that a significant number of couplings involve putative nonhomotypic channels. (+info)
Exchange of conductance and gating properties between gap junction hemichannels.
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Gap junction channels span the membranes of two adjacent cells and allow the gated transit of molecules as large as second messengers from cell to cell. The structure of the gap junction channel pore is not resolved. For identification of pore determinants we used a chimera of two connexins, cx46 and cx32E(1)43, that form membrane channels with distinct unit conductances and channel kinetics. Exchange of the first transmembrane segment (M1) between these connexins resulted in a chimera that exhibited most of the channel properties of the M1 donor, including single channel conductance, channel kinetics, and the preference to dwell at a subconductance level. The M1 segment thus appears to be an important determinant of conductance and gating properties of connexin channels. (+info)