Structure of the Agonist-Binding Sites of the Torpedo Nicotinic Acetylcholine Receptor: Affinity-Labeling and Mutational Analyses Identify γTyr-111/δArg-113 as Antagonist Affinity Determinants

Publisher Website
Google Scholar
Abstract
Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the γ-subunit which, along with the analogous residue in δ-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and α-conotoxin (αCtx) MI. nAChR γ-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in γTyr-111 and γTyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at γTrp-55, the primary site of [3H]dTC photoincorporation within γ-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940−32950]. The Torpedo nAChR δ-subunit residue corresponding to γTyr-111 (δArg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the α−δ agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]α-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and αCtx MI with nAChRs containing γY111R or δR113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the γY111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of αCtx MI, with δR113Y enhancing and γY111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of γ- (or δ-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.