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

Abstract

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-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 gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.

Site-specific Charge Interactions of α-Conotoxin MI with the Nicotinic Acetylcholine Receptor

“…Some of the larger differences can be accounted for by the distinct buffer conditions used as follows: Hann et al (13) and Groebe et al (10) used low ionic strength buffers to measure binding in the presence of detergents. However, the data of Chiara et al (12) was carried out in physiological buffer that revealed affinities 5-10-fold higher than we observed. Our binding data on the mouse muscle AChR is in complete agreement with that of others (7).…”

“…At the Torpedo AChR ␦-subunit, acetylation of Lys-10 does appear to cause an increase in affinity of 2-4-fold, consistent with lessened repulsion. However, this change is much smaller than the 1000-fold effect observed by Chiara et al (12). Our data suggest that the ␦Arg-113 interaction is not through electrostatic repulsion at the N terminus, His-5, or Lys-10 of Ctx.…”

“…The homologous residue to ␥Tyr-111 in Torpedo ␦-subunit is ␦Arg-113. Chiara et al (12) demonstrated that ␦Arg-113 dramatically weakened binding of Ctx to the ␣␦ site, and they inferred an electrostatic repulsion with Lys-10. At the Torpedo AChR ␦-subunit, acetylation of Lys-10 does appear to cause an increase in affinity of 2-4-fold, consistent with lessened repulsion.…”

“…Significant affinity differences have also been observed for binding to the Torpedo AChR (10 -12); however, in this case the ␣␥ site is bound with higher affinity. Chiara et al (12) showed that one AChR residue, ␥Tyr-111, and its ␦-subunit homolog, ␦Arg-113, accounted for much of the affinity differences in Torpedo AChR. They further suggested that the affinity change arose from specific charge repulsion between ␦Arg-113 and Ctx residue Lys-10.…”

The Role of Tyrosine Residues in the Extracellular Domain of the 5-Hydroxytryptamine3 Receptor

“…Tyr 141 , Tyr 143 , and Tyr 153 align with residues in the nACh receptor binding loop E. Extensive studies on the nACh receptor and the ␥-aminobutyric acid A receptor have indicated the importance of residues within this binding loop as determinants of antagonist affinity (31)(32)(33)(34)(35)(36). The lack of sequence conservation in this region in particular is thought to confer the differences in antagonist selectivity.…”