Abstract

We investigated the molecular nature of the interaction between the functionally selective M₁ muscarinic acetylcholine receptor (mAChR) agonist xanomeline and the human M₁ mAChR expressed in Chinese hamster ovary (CHO) cells. In contrast to the non-subtype-selective agonist carbachol, xanomeline demonstrated M₁ mAChR binding that was resistant to extensive washout, resulting in a significant reduction in apparentN-[³H]methylscopolamine saturation binding affinity in intact cells. Functional assays, using both M₁mAChR-mediated phosphoinositide hydrolysis and activation of neuronal nitric oxide synthase, confirmed that this persistent binding resulted in elevated basal levels of system activity. Furthermore, this phenomenon could be reversed by the addition of the antagonist atropine. However, pharmacological analysis of the inhibition by atropine of xanomeline-mediated functional responses indicated a possible element of noncompetitive behavior that was not evident in several kinetic and equilibrium binding experimental paradigms. Taken together, our findings indicate for the first time a novel mode of interaction between an mAChR agonist and the M₁ mAChR, which may involve unusually avid binding of xanomeline to the receptor. This yields a fraction of added agonist that is retained at the level of the receptor compartment to persistently bind to and activate the receptor subsequent to washout. The results of the current study suggest that elucidation of the mechanism or mechanisms of interaction of xanomeline with the M₁ mAChR is particularly important in relation to the potential therapeutic use of this agent in the treatment of Alzheimer’s disease.