The M4 Helix Is Involved in α7 nACh Receptor Function

Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1-M4) with M2 lining the pore and then M1 and M3, with M4 outermost and adjacent to the membrane lipids. M4 has a variety of roles: its interaction with neighboring M1 and M3 helices is important for receptor assembly, it can a transmit information on the lipid content of the membrane to the gating mechanism, and it may form a vital link to the extracellular domain via the Cys-loop. This study examines the role of M4 receptor residues in the α7 nAChR using site-directed mutagenesis and subsequent expression in Xenopus oocytes. The data indicate that many of the residues in M4 play a role in receptor function, as substitution with Ala can modify functional parameters; 11 of 24 mutants showed a small gain of function (<10-fold decrease in EC₅₀), and 1 (D446A) did not respond to the agonist; it was also not expressed at the cell surface. Removal or addition of aromatic residues had small or no effects. These results demonstrate the α7 nAChR M4 has a role in receptor function, and a structural model suggests possible interactions of some of these residues with their neighbors.