Atypical Muscarinic Allosteric Modulation: Cooperativity between Modulators and Their Atypical Binding Topology in Muscarinic M2 and M2/M5 Chimeric Receptors

The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are “atypical”, having steep concentration-effect curves and not interacting competitively with “typical” allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M 2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4′-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1′-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg 2+ , and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M 2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M 2 /M 5 -receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M 2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the “common” allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M 2 -receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer.