Residues at the Subunit Interfaces of the Nicotinic Acetylcholine Receptor That Contribute to α-Conotoxin M1 Binding

Abstract

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition α₂βγδ are formed by nonequivalent α-γ and α-δ subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that ¹²⁵I-α-conotoxin M1 binds with high affinity to the α-δ subunit dimers, but not to α-γ dimers, nor to α, γ, and δ monomers, a finding consistent with α-conotoxin M1 selectivity for the αδ interface in the intact receptor measured by competition against α-bungarotoxin binding. We also extend previous identification of α-conotoxin M1 determinants in the γ and δ subunits to the α subunit interface by mutagenesis of conserved residues in the α subunit. Most mutations of the α subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant α and non-α subunits, indicating that side chains of the α subunit do not interact with those of the γ or δ subunits in stabilizing α-conotoxin M1. The overall findings suggest different binding configurations of α-conotoxin M1 at the α-δ and α-γ binding interfaces.