Characterization of d-tubocurarine binding site of Torpedo acetylcholine receptor

d-Tubocurarine (curare) is a well-characterized competitive antagonist of nicotinic acetylcholine receptors (AChRs), and it is usually assumed that curare and agonists share a common binding site. We have examined the role of several highly conserved residues of the α-, γ-, and δ-subunits in the interaction of curare with the Torpedo acetylcholine receptor (AChR). Curare inhibition of wild-type receptors is consistent with curare binding to a single high-affinity binding site [inhibitor constant (K(i)) = 20 nM]. Phenylalanine substitutions for two tyrosine residues implicated as being in the ligand binding site (αY93F, αY190F) reduce curare affinity, indicating that these residues are also important for high-affinity curare binding. Phenylalanine substitution for αY198 [αY198F (notation used here: subunit/amino acid in wild-type/residue number/substitution)] causes a 10- fold increase in curare affinity (K(i) = 3.1 nM), and measurement of the recovery from curare inhibition indicates that this increase in affinity is due to a reduction in the rate of curare dissociation from the receptor. In addition to the α-subunits, portions of the ligand binding sites also reside on the γ- and δ-subunits, and photoaffinity studies have implicated two residues (γW55 and δW57) as forming part of the curare sites. The γW55L mutation results in an eightfold decrease in curare affinity (K(i) = 170 nM), whereas the δW57L mutation has no effect. These data support the notion that the high-affinity curare binding site is formed by segments of the α- and γ-subunits. Most of the mutations that affect ACh activation have a similar effect on curare inhibition, suggesting that agonists and antagonists interact with many of the same amino acid residues in the binding site.