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 assum ed that curare and agonists share a common binding site. We have exami ned the role of several highly conserved residues of the alpha-, gamma -, and delta-subunits in the interaction of curare with the Torpedo ac etylcholine receptor (AChR). Curare inhibition of wild-type receptors is consistent with curare binding to a single high-affinity binding si te [inhibitor constant (K-i) = 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 thes e residues are also important for high-affinity curare binding. Phenyl alanine 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 (K-i = 3.1 nM), and measurement o f 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 oc-subunits, portions of the ligand binding sites also reside on the gamma- and delta-subunits, and photoa ffinity 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 (K-i = 170 nM), whereas th e delta W57L mutation has no effect. These data support the notion tha t the high-affinity curare binding site is formed by segments of the a lpha- and gamma-subunits. Most of the mutations that affect ACh activa tion have a similar effect on curare inhibition, suggesting that agoni sts and antagonists interact with many of the same amino acid residues in the binding site.