Abstract

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M₃receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M₂ 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) and the negative effect of gallamine on the affinity for classical antagonistN-[³H]methylscopolamine ([³H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M₃ receptors was made identical with the o3 loop of M₂ receptors, and alcuronium acquired positive influence on the affinity for [³H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M₂ to M₃ receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [³H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M₃ receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [³H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [³H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M₂ 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.