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

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Abstract
The effects on the whole‐cell carbachol‐induced muscarinic cationic current (mIcat) of antibodies against the α‐subunits of various G proteins, as well as the effect of a Gβγ 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. Application of ascending concentrations of carbachol (1–300 μM) activated mIcat (mean amplitude 0.83 nA at 300 μM carbachol; EC50 8 μM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of Gi3/Go or Go antibodies resulted in about a 70% depression of the maximum response without change in the EC50 value. In contrast, antibodies against α‐subunits of Gi1, Gi1/Gi2, Gi3, Gq/G11 or Gs protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gβ or infusion of the Gβγ subunit itself had no effect on mIcat. If cells were exposed briefly to carbachol (50 or 100 μM) at early times (αi3/Gαo or to Gαo had begun, carbachol responses remained unchanged even after 20–60 min; that is, the depression of mIcat by these antibodies was prevented. These data show that Gαo 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. British Journal of Pharmacology (2003) 139, 605–615. doi:10.1038/sj.bjp.0705289