Scanning mutagenesis identifies amino acid side chains in transmembrane domain 5 of the M(1) muscarinic receptor that participate in binding the acetyl methyl group of acetylcholine.

The exofacial part of transmembrane domain 5 (TMD 5) of the cationic amine-binding subclass of 7-transmembrane receptors is thought to be important in binding the side chain of the agonist. Residues Ile-188 through Ala-196 in TMD 5 of the M(1) muscarinic acetylcholine receptor (mAChR) have been studied by Cys- and Ala-scanning mutagenesis. The results are consistent with a helical conformation for this sequence. The positively charged sulfhydryl reagent N-trimethyl-2-aminoethyl methanethiosulfonate reacted selectively with Phe-190 --> Cys, Thr-192 --> Cys, and Ala-193 --> Cys, indicating that the face of TMD 5 accessible from the binding site crevice is consistent with a recent model by Baldwin and colleagues of the transmembrane domain of the 7-transmembrane receptors. In contrast, the acetylcholine derivative bromoacetylcholine reacted selectively with Thr-192 --> Cys, which forms the focus of a group of amino acids (Ile-188, Thr-189, Thr-192, Ala-196) whose mutation decreased the binding affinity of the transmitter ACh itself. The center of this patch of residues is offset to one side of the binding pocket, suggesting that a rotation of TMD 5, relative to that implied by the Baldwin model, may be necessary to optimize the anchoring of acetylcholine within the binding site of the M(1) mAChR. An induced rotation of TMD 5 could contribute to the formation of the activated state of the receptor.