Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M-3 receptors

To clarify the involvement of specific domains of muscarinic receptors in t he action of allosteric modulators, muscarinic M-3 receptors (on which allo steric interactions are weak) were genetically modified to become more simi lar to M-2 receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) an d the negative effect of gallamine on the affinity for classical antagonist N-[H-3]methylscopolamine ([H-3]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M-3 receptors was made identical with the o3 loop of M-2 receptors, and alcuronium acquired positive influe nce on the affinity for [H-3]NMS. This is the first instance of inducing po sitive cooperativity on muscarinic receptors by genetic manipulation. Trans ferring whole o2 loop from M-2 to M-3 receptors substantially enhanced affi nities for gallamine and alcuronium without augmenting their negative actio n on [H-3]NMS binding. In contrast, effects of simply adding two negative c harges into the o2 loop of M-3 receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [ H-3]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [H-3]NM S and alcuronium and gallamine, respectively, and in the binding of both mo dulators to M-2 receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.