Sine_1994_J.Biol.Chem_269_8808

Studies with site-directed labeling reagents have identified residues near the ligand binding pocket of the nicotinic acetylcholine receptor. Among these residues are three conserved tyrosines, Tyr-93, Tyr-190, and Tyr-198 of the alpha subunit. Previous studies combined mutagenesis, expression in Xenopus oocytes, and dose-response analysis to examine contributions of these tyrosines to agonist affinity. In this study, we prepared a series of mutants at each position, expressed them in 293 HEK cells, and studied binding of agonists and antagonists to mutant receptors on intact cells. We show that all three tyrosines contribute to binding of agonists, and that each tyrosine contributes roughly equally to the binding energy. Although the contributions are roughly equivalent, the nature of the contribution is not equivalent at each position. For Tyr-93 and Tyr-190 the aromatic hydroxyl is essential, whereas for Tyr-198 aromaticity of the side chain is essential. Nearly identical results were obtained for the elementary quaternary ligand tetramethylammonium, indicating that these tyrosines contribute to stabilization of the quaternary ammonium portion of agonist. Tyr-190 and Tyr-198 also contribute to binding of the competitive antagonist dimethyl-d-tubocurarine; the side chain specificity for binding supports tyrosine interactions with one of two quaternary ammonium groups in dimethyl-d-tubocurarine. Y190F, in addition to altering binding affinity, also affects the equilibrium between activatable and desensitized receptor states.