MOLECULAR DISSECTION OF SUBUNIT INTERFACES IN THE ACETYLCHOLINE-RECEPTOR - IDENTIFICATION OF DETERMINANTS OF ALPHA-CONOTOXIN M1 SELECTIVITY

The acetylcholine receptor from vertebrate skeletal muscle is a pentam er of homologous subunits with composition alpha(2) beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta in terfaces, differ in their affinities for agonists and competitive anta gonists, owing to different contributions of the gamma and delta subun its. To identify portions of the gamma and delta subunits that contrib ute to the binding sites, the experiments described here use gamma-del ta subunit chimeras and site-specific mutants to determine the basis o f the 10,000-fold selectivity of conotoxin M1 for the sites. Three dis tinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible far the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, a nd I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limite d model of the folding pattern of the extracellular domain of the subu nits.