CONSERVED TYROSINES IN THE ALPHA-SUBUNIT OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR STABILIZE QUATERNARY AMMONIUM GROUPS OF AGONISTS AND CURARIFORM ANTAGONISTS

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 mutagenes is, expression in Xenopus oocytes, and dose-response analysis to exami ne 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 muta nt receptors on intact cells. We show that all three tyrosines contrib ute 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, w hereas for Tyr-198 aromaticity of the side chain is essential. Nearly identical results were obtained for the elementary quaternary ligand t etramethylammonium, indicating that these tyrosines contribute to stab ilization of the quaternary ammonium portion of agonist. Tyr-190 and T yr-198 also contribute to binding of the competitive antagonist dimeth yl-d-tubocurarine; the side chain specificity for binding supports tyr osine interactions with one of two quaternary ammonium groups in dimet hyl-d-tubocurarine. Y190F, in addition to altering binding affinity, a lso affects the equilibrium between activatable and desensitized recep tor states.