The functional topography of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor, revealed by scanning mutagenesis

Alanine-scanning mutagenesis has been applied to residues 100-121 in transmembrane domain 3 of the M-1 muscarinic acetylcholine receptor, This study complements a previous investigation of the triad Asp(122)-Arg(123)-Tyr(124) (Lu, Z-L., Curtis, C, A,, Jones, P. G,, Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol 51, 234-241), The results demonstrate the alpha-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val(113), Leu(116), and Ser(120), which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G; protein) or intrareceptor beads that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second.