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 alpha-, gamma-, and delta-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 (Ki) = 20 nM]. Phenylalanine 
substitutions for two tyrosine residues implicated as being in the ligand 
binding site (alpha Y93F, alpha Y190F) reduce curare affinity, indicating that 
these residues are also important for high-affinity curare binding. 
Phenylalanine substitution for alpha Y198 [alpha Y198F (notation used here: 
subunit/amino acid in wild-type/residue number/substitution)] causes a 10-fold 
increase in curare affinity (Ki = 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 
alpha-subunits, portions of the ligand binding sites also reside on the gamma- 
and delta-subunits, and photoaffinity studies have implicated two residues 
(gamma W55 and delta W57) as forming part of the curare sites. The gamma W55L 
mutation results in an eightfold decrease in curare affinity (Ki = 170 nM), 
whereas the delta W57L mutation has no effect. These data support the notion 
that the high-affinity curare binding site is formed by segments of the alpha- 
and gamma-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)