Abstract

Site-directed mutagenesis and expression in Xenopus oocytes were used to study acetylcholine receptors in which serine residues (i) were replaced by alanines (α, δ subunits) or (ii) replaced a phenylalanine (β subunit) at a postulated polar site within the M2 transmembrane helix. As the number of serines decreased, there were decreases in the residence time and consequently the equilibrium binding affinity of QX-222, a quaternary ammonium anesthetic derivative thought to bind within the open channel. Receptors with three serine-to-alanine mutations also displayed a selective decrease in outward single-channel currents. Both the direction of this rectification and the voltage dependence of QX-222 blockade suggest that the residues mutated are within the aqueous pore of the receptor and near its cytoplasmic (inner) surface.