Critical residues influence the affinity and selectivity of α-conotoxin MI for nicotinic acetylcholine receptors

The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the R/‰ and R/A subunit interfaces. R-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has 10 4 higher affinity for the R/‰ site. We performed an "alanine walk" to identify the residues in R-MI that contribute to this selective interaction with the R/‰ site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the A or ‰ subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity R/‰ site while having comparatively little effect on low-affinity R/A binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45 000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the R/‰ site without affecting R/A binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the R/‰ site but also increases by 500-fold at the R/A site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity R/‰ binding site.