We have tested the importance of charge interactions for ␣-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on ␣-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the ␣␦ site but not at the ␣␥ site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the ␣␦ site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, ␣-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscletype nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR ␥-subunit. Because data for mutations at residue ␥Tyr 117 were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of ␥Tyr