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 gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) 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 gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi 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 gammaTyr(117). 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.
Highlights
The muscle nicotinic acetylcholine receptor (AChR)[1] is a member of the ligand-gated ion channel superfamily
In order to examine the proximal relationship of AChR amino acid residues to functional groups on dTC, we assessed the degree of interaction by double mutant thermodynamic cycle analysis
The affinities of mutant and wild type AChRs were measured for a pair of dTC analogs
Summary
The muscle nicotinic acetylcholine receptor (AChR)[1] is a member of the ligand-gated ion channel superfamily. Identification of binding site residues has been carried out by affinity and photoaffinity labeling, by cross-linking studies, by analysis of expressed chimeric receptors, and by site-directed mutagenesis (5) These approaches have found that the binding sites consist of residues from two subunits from several regions ( referred to as loops) that are distant in the linear subunit sequences. The prevalence of aromatic amino acids as well as detailed studies using unnatural amino acid substitution support the hypothesis that cation -electron interaction are critical for stabilizing the ammoniums of agonists (11, 17) Such studies yielded observations on the possible interactions of several other aromatic residues; Nowak et al (17) showed that ␣Tyr[93] is likely to act as a hydrogen bond donor, whereas the aromatic ring of ␣Tyr[198] appears to interact with the quater-
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