Abstract

Transmembrane domain 6 of the muscarinic acetylcholine (ACh) receptors is important in ligand binding and in the conformational transitions of the receptor but the roles of individual residues are poorly understood. We have carried out a systematic alanine-scanning mutagenesis study on residues Tyr381 to Val387 within the binding domain of the M₁ muscarinic ACh receptor. The seven mutations were then analyzed to define the effects on receptor expression, agonist and antagonist binding, and signaling efficacy. Tyr381Ala produced a 40-fold reduction in ACh affinity and a 50-fold reduction in ACh-signaling efficacy. Leu386Ala had similar but smaller effects. Asn382Ala caused the largest inhibition of antagonist binding. The roles of the hydroxyl group and benzene ring of Tyr381 were probed further by comparative analysis of the Tyr381Phe and Tyr381Ala mutants using three series of ligands: ACh analogs, azanorbornane- and quinuclidine-based ligands, and atropine analogs. These data suggested that the hydroxyl group of Tyr381 is primarily involved in forming hydrogen bond interactions with the oxygen atoms present in the side chain of ACh. We propose that this interaction is established in the ground state and preserved in the activated state of the receptor. In contrast, the Tyr381 benzene ring may form a cation-π interaction with the positively charged head group of ACh that contributes to the activated state of the receptor but not the ground state. However, the hydroxyl group and benzene ring of Tyr381 both participate in interactions with azanorbornane- and quinuclidine-based ligands and atropine analogs in the ground state as well as the activated state of the receptor.