Effects of G-protein-specific antibodies and Gβγ subunits on the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells

1 The effects on the whole-cell carbachol-induced muscarinic cationic current (mIcat) of antibodies against the alpha-subunits of various G proteins, as well as the effect of a Gbetagamma subunit, were studied in single guinea-pig ileal smooth muscle cells voltage-clamped at -50 mV. Ionized intracellular calcium concentration, [Ca2+](i), was clamped at 100 nM using a 1,2-bis(2-aminophenoxyl- ethane-N,N,N',N'-tetraacetic acid)/Ca2+ mixture. 2 Application of ascending concentrations of carbachol (1-300 muM) activated mIcat (mean amplitude 0.83 nA at 300 muM carbachol; EC50 8 muM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of G(i3)/G(o) or G(o) antibodies resulted in about a 70% depression of the maximum response without change in the EC50 value. In contrast, antibodies against alpha-subunits of G(i1), G(i1)/ G(i2), G(i3), G(q)/ G(11) or G(s) protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gbeta or infusion of the Gbetagamma subunit itself had no effect on mIcat. 3 If cells were exposed briefly to carbachol (50 or 100 muM) at early times (<3 min) after infusion of antibodies to Gα(i3)/Gα(o) or to Gα(o) had begun, carbachol responses remained unchanged even after 20 - 60 min; that is, the depression of mIcat by these antibodies was prevented. 4 These data show that Gα(o) protein couples the muscarinic receptor to the cationic channel in guinea-pig ileal longitudinal smooth muscle and that Gβγ is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long-lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.