The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition α2βγδ. Its two ligand binding sites, formed at α-γ and α-δ interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the γ and δ subunits. To identify portions of the † and δ subunits that contribute to the binding sites, the experiments described here use γ-δ subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the γ subunit confer low affinity to the α-γ binding site, whereas the corresponding residues of the δ subunit, S36, Y113, and 1178, confer high affinity to the α-δ site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.
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