RESIDUES AT THE SUBUNIT INTERFACES OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR THAT CONTRIBUTE TO ALPHA-CONOTOXIN M1 BINDING

The two binding sites in the pentameric nicotinic acetylcholine recept or of subunit composition alpha(2) beta gamma delta are formed by non- equivalent alpha-gamma and alpha-delta subunit interfaces, which produ ce site selectivity in the binding of agonists and antagonists. We sho w by sedimentation analysis that I-125-alpha-conotoxin M1 binds with h igh affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit int erface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two s ites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn aff ect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subun its, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotox in M1. The overall findings suggest different binding configurations o f alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interf aces.