GTP-binding protein Gq mediates muscarinic-receptor-induced inhibition of the inwardly rectifying potassium channel IRK1 (Kir 2.1).
The inwardly rectifying potassium channel IRK1, a member of the Kir 2.0 family, is inhibited by m1 muscarinic receptor stimulation. In this study the mechanism of action underlying the muscarinic response was investigated by identification of the subtype of heterotrimeric G-protein involved in transduction of the signal. tsA201 cells were simultaneously transfected with cDNAs encoding IRK1, m1 and the Galpha subunit of either G(q), G(12) or G(13). The whole-cell patch-clamp technique was used to study the effects of G-protein transfection. Antibodies generated against the C-terminal regions of Galpha(q/11) and Galpha(12) were used to confirm G-protein expression by Western blot. When challenged with carbachol, IRK1 currents recorded from cells co-transfected with Galpha(q) were potently inhibited compared with controls. Conversely, co-transfection with Galpha(12) or Galpha(13) subunits had no effect on muscarinic-receptor-induced inhibition of IRK1. Concentration response curves revealed that carbachol was 16 times more potent at inhibiting IRK1 currents in cells co-transfected with Galpha(q) as compared with Galpha(12) co-transfected cells. Immunoblotting illustrated low levels of endogenous Galpha(q/11) and Galpha(12) in untransfected tsA cells. Transfection with Galpha(q) or Galpha(12) cDNAs greatly increased the levels of G-protein expression in both cell populations. G-protein expression did not interfere with m1 muscarinic receptor expression levels. These findings suggest that the m1 muscarinic-receptor-induced inhibition of IRK1 is mediated by the heterotrimeric G-protein, Galpha(q), in tsA cells.
Firth
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TA
Department of Psychiatry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Jones
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SV
D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D002217:Carbachol; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins