Functional analysis of transmembrane domain 2 of the M1 muscarinic acetylcholine receptor

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M-1 muscarinic acetylcholine receptor, and of the highly conserved Asn(43) in TM1. The mutation of Asn(43), Asn(61), and Leu(64) caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro(415) and Tyr(418) in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp(71) and Asn(414) may be a critical feature stabilizing the activated state of the M-1 receptor. Mutation of Leu(67), Ala(70), and Ile(74) also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp(71) and Asn(414). Mutation of Leu(72), Gly(75), and Met(79) in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met(79) as well as Asp(71).