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Structural Requirements of Transmembrane Domain 3 for Activation by the M₁ Muscarinic Receptor Agonists AC-42, AC-260584, Clozapine, and N-Desmethylclozapine: Evidence for Three Distinct Modes of Receptor Activation

ACADIA Pharmaceuticals, Inc., San Diego, California

Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M₁ muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M₁. In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M₁ but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M₁ receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.

Address correspondence to: Dr. Ethan S. Burstein, ACADIA Pharmaceuticals, Inc., 3911 Sorrento Valley Boulevard, San Diego, CA 92121. E-mail: eburstein{at}acadia-pharm.com

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