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

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 G βγ subunit, were studied in single guinea‐pig ileal smooth muscle cells voltage‐clamped at −50 mV. Ionized intracellular calcium concentration, [Ca 2+ ] i , was clamped at 100 n M using a 1,2‐bis(2‐aminophenoxyl‐ethane‐ N , N , N ′, N ′‐tetraacetic acid)/Ca 2+ mixture. Application of ascending concentrations of carbachol (1–300 μ M ) activated mIcat (mean amplitude 0.83 nA at 300 μ M carbachol; EC 50 8 μ M ; 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 EC 50 value. In contrast, antibodies against α ‐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 G β or infusion of the G βγ subunit itself had no effect on mIcat. If cells were exposed briefly to carbachol (50 or 100 μ M ) at early times (<3 min) after infusion of antibodies to G α i3 /G α o or to G α o 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 G α o protein couples the muscarinic receptor to the cationic channel in guinea‐pig ileal longitudinal smooth muscle and that G βγ 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