Exploring the Mechanism of Agonist Efficacy: A Relationship between Efficacy and Agonist Dissociation Rate at the Muscarinic M3

Although there are several empirical approaches that enable the comparison of relative agonist efficacy, the molecular basis that underlies differences in the ability of G protein-coupled receptor agonists to elicit a response is still largely unexplained. Several models have been described that incorporate the ki-netics of receptor-mediated initiation of the G protein cycle, but these have not directly addressed the influence of agonist-binding kinetics. To test this, we investigated the relationship between the efficacy of seven M3 muscarinic receptor agonists and their rate of dissociation (koff) from the M3 receptor. The association and dissociation rate constants of the agonists were determined using a l-[N-methyl]-[3H]scopolamine methyl chloride competition binding assay in the presence of GTP. The agonists displayed a range of association and dissociation rates. Relative agonist efficacy was measured at two points after M3 receptor activation: the stimulation of guanosine 5-O-(3-[35S]thio)triphosphate binding to G subunits, and the sub-sequent increase in intracellular calcium levels. These experi-ments revealed a range of intrinsic efficacy, from the low-efficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist effi-cacy and the equilibrium binding affinity of each agonist (Kd). When efficacy was compared with the dissociation rate con-stant, however, the two were highly correlated, suggesting a relationship between the duration of agonist binding at the receptor and the intrinsic efficacy. These data suggest that kinetic models incorporating the mean lifetime of specific com-plexes will be required to fully explain the nature of agonist efficacy. Efficacy is defined as the ability of a ligand to elicit a response upon binding to a receptor (Stephenson, 1956) and is arguably the most important parameter for optimization in novel agonist drugs. To date, efficacy has largely been treated as an empirical term, and several approaches comparing equilibrium binding to functional potency have been de