Chloride channels in myocytes from rabbit colon are regulated by a pertussis toxin-sensitive G protein.
Because of the high intracellular Cl- concentration ([Cl-]i) in gastrointestinal smooth muscle, receptor-mediated opening of Cl- channels at the cell resting potential could represent a plausible mechanism for initial receptor-mediated cell depolarization. To test this hypothesis, we characterized activation of large-conductance Cl- channels by the neurokinin-1 (NK-1) receptor agonist [Sar9,Met(O2)11]-substance P, by specific second messengers, and by direct G protein activation in myocytes isolated from the rabbit colon longitudinal muscle layer. In excised inside-out patches, large-conductance ion channels selective for Cl- over Na+ could be induced by holding the patch at pipette potentials values > 60 mV. The channel showed multiple smaller conductance states (< or = 20) but could open and close via a main gate. When the channel was fully open, its slope conductance was 300 pS, with substates as small as 15 pS, comparable to the predominant conductance observed in cell-attached patches. The voltage-activation profile for full conductance was bell-shaped with maximal open probability (Po) for channel opening of approximately 0 mV. In cell-attached patches, addition of the NK-1 agonist to pipette solution activated a channel that corresponded to a subconductance state of the maxi Cl- channel. The voltage-activation profile for this subconductance state showed a maximal Po value for membrane potentials of approximately 0 mV, with rapid inactivation at more positive and partial inactivation at more negative membrane potentials. In excised inside-out patches, both the full and smaller conductance states of the Cl- channel were activated by the nonhydrolyzable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) and inhibited by pertussis toxin (PTX), whereas [Ca2+]i increased channel activity only in concentrations > 1 mM. In cell-attached patches, addition of different Ca2+ ionophores resulted in channel activation in 10% of cells, and activators of protein kinase A or protein kinase C had no effect. These findings are consistent with the hypothesis of a possible role of G protein-coupled Cl- channels in receptor-mediated initial cell depolarization in longitudinal colonic smooth muscle.
Sun
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Department of Medicine, Veteran's Affairs Wadsworth Medical Center, Los Angeles 90073.
Supplisson
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Mayer
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D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D004553:Electric Conductivity; D004594:Electrophysiology / Q000379:methods; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D008536:Meglumine / Q000494:pharmacology; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology*; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011817:Rabbits; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000502:physiology; D013373:Substance P / Q000031:analogs & derivatives / Q000494:pharmacology; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D018118:Chloride Channels; D002712:Chlorides; D004075:Diglycerides; D007473:Ion Channels; D008565:Membrane Proteins; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; D013757:Tetraethylammonium Compounds; D010566:Virulence Factors, Bordetella; D009243:NAD; C062618:substance P, Sar(9)-Met(O2)(11)-; D005576:Colforsin; D013373:Substance P; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000001:Calcimycin; D015759:Ionomycin; D003994:Bucladesine; D019789:Tetraethylammonium; D008536:Meglumine; C038237:1-oleoyl-2-acetylglycerol; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins