Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site

Abstract

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type 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 γ-subunit. Because data for mutations at residue γTyr117 were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of γTyr117 with both the 2-N and the 13′-positions of dTC. Examination of a dTC analog with a negative charge at the 13′-position failed to reveal electrostatic interaction with charged side-chain substitutions at γ117, but the effects of side-chain substitutions remained consistent with proximity of Tyr117 to the cationic 2-N of dTC. The apparent interaction of γTyr117 with the 13′-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-π electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to γTyr117. This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.