• Smooth muscle;
  • GTP binding protein;
  • muscarinic receptor;
  • carbachol;
  • cationic current;
  • antibody
  • 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 (<3 min) after infusion of antibodies to Gαi3/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