This amended competing renewal for years 15-18 aims at investigating the mechanisms of the unconventional mode of activation of muscarinic acetylcholine receptors by the muscarinic receptor agonist, xanometine, in cultured cerebral cortical neurons. Xanomeline demonstrates unique functional selectivity in activating M1 muscarinic receptors that are important for learning ad memory. Thus, xanomeline-like muscarinic agonists are considered promising candidates for drug development for cholinergic replacement therapy in Alzeimer's disease. During the current funding period, we serendipitousiy discovered that xanomeline activates M1 muscarinic receptors in a long-lasting and wash-resistant manner. Moreover, we obtained strong evidence that this novel property of xanomeline is not due to an experimental artifact or nonspecific interaction with the cell membrane, but is rather the result of its avid binding to a specific and saturable site located on the M1 muscarinic receptor. We have also obtained preliminary evidence using pharmacology and computer modeling of receptor structure that this persistent interaction of xanometine with the M1 muscarinic receptor takes place at an """"""""exosite"""""""" that is distinct from where conventional agonists bind. The proposed project aims at applying a combination of pharmacologic tools, computer modeling, sitedirected mutagenesis and structure-activity relationship studies in an iterative manner to delineate the molecular nature of the muscarinic receptor exosite and the interaction of xanomeline at that site. These studies are expected to have significant impact on the development of additional muscarinic receptor agonists that exhibit long-lasting receptor activation for therapy of Alzheimer's dementia. They may also illustrate novel mechanisms of activation of muscarinic receptors that might be applicable to other members of the G protein coupled receptor family.
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