Tota_1990_Mol.Pharmacol_37_996

The mechanism of action of the partial muscarinic agonist pilocarpine was analyzed in a reconstituted system consisting of the purified porcine atrial muscarinic receptor and the purified porcine atrial inhibitory guanine nucleotide-binding protein, Gi. When GTPase activity was measured as a function of receptor.agonist complex concentration at saturating concentrations of either the full agonist carbachol or pilocarpine, both ligands gave similar values of kcat (4.3 +/- 0.2 min-1 for carbachol and 5.4 +/- 0.7 min-1 for pilocarpine); however, the observed dissociation constant for the ligand.receptor complex binding to Gi was about 4-fold lower for carbachol (0.81 +/- 0.19 nM) than for pilocarpine (3.02 +/- 0.83 nM). These results suggested that, in this system, the reduced activity of the partial agonist compared with the full agonist was the result of a decrease in affinity of the receptor.ligand complex for Gi, as opposed to differences in their relative abilities to activate the guanine nucleotide-binding protein. Several analogues of oxotremorine were also tested to determine their effects on the GTPase activity of Gi. Results from these studies indicate that the reconstituted system may be useful in determining structure-function relationships for muscarinic agonists with regard to receptor.Gi interactions.