Muscarinic inhibition of single KCa channels in smooth muscle cells by a pertussis-sensitive G protein.
Application of a muscarinic agonist to the extracellular surface of membrane patches from airway smooth muscle cells resulted in an inhibition of calcium-activated potassium (KCa) channels in outside-out patches. Methacholine (50 microM) inhibited channel activity at physiological cytosolic calcium concentrations and resulted in a marked shift in channel open-time kinetics. In inside-out patches, KCa channels were inhibited upon addition of GTP (100 microM) when methacholine was present in the patch pipette. Muscarinic inhibition was blocked when guanosine 5'-O-(2-thiodiphosphate) was used to compete with endogenous GTP in outside-out or inside-out experiments. Pretreatment of dissociated cells with pertussis toxin (0.1 micrograms/ml) blocked muscarinic inhibition of the channel in a time-dependent fashion. These results demonstrate, at the single-channel level, a coupling between muscarinic receptor stimulation and inhibition of KCa in smooth muscle and demonstrate the guanine nucleotide dependence of this coupling.
Kume
H
H
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104.
Kotlikoff
M I
MI
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004285:Dogs; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials; D016210:Methacholine Chloride / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D037342:Pertussis Toxin; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D015221:Potassium Channels; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D016210:Methacholine Chloride; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D011188:Potassium; D002118:Calcium