M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor

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, then M1 and M3, and with M4 outermost and adjacent to the membrane lipids. Despite its remote location, M4 contributes both to receptor assembly and gating in pLGICs where it has been examined. This study probes the role of M4 residues in the α4β2 nAChR using site-directed mutagenesis to individually mutate each residue to alanine, followed by expression in HEK293 cells and then characterization using membrane potential sensitive dye and radioligand binding. Two of the resulting mutant receptors showed altered EC₅₀s, while 13 were nonfunctional, although coexpression with the chaperones RIC3 and nAChO resulted in 4 of these responding to agonist. Of the remaining 9, radioligand binding with epibatidine showed that 8 were expressed, suggesting these residues may play a role in channel opening. These data differ from similar studies in other pLGIC, where few or no Ala mutants in M4 ablate function, and they suggest that the α4β2 nAChR M4 may play a more significant role than in related receptors.