Molecular dissection of subunit interfaces in the acetylcholine receptor: Identification of residues that determine curare selectivity

The acetylcholine receptor from vertebrate skeletal muscle is a transmembrane channel that binds nerve-released acetylcholine to elicit rapid transport of small cations. Composed of two α subunits and one β, one γ, and one δ subunit, the receptor is a cooperative protein containing two sites that bind agonists, curariform antagonists, and snake α-toxins. Until recently the two binding sites were thought to reside entirely within each of the two α subunits, but affinity labeling and expression studies have demonstrated contributions by the γ and δ subunits. Affinity labeling and mutagenesis studies have identified residues of the α subunit that contribute to the binding site, but the corresponding γ- and 5-subunit residues remain unknown. By making γ-δ chimeras and following the nearly 100-fold difference in curare affinity for the two binding sites, the present work identified residues of the γ and δ subunits likely to be near the binding site. Two sets of binding determinants were identified in homologous positions of the γ and δ subunits. The determinants lie on either side of a disulfide loop found within the major extracellular domain of the subunits. This loop is common to all acetylcholine, γ-aminobutyrate, and glycine receptor subunits.