Identification of amino acids of the torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [3H]tetracaine

[H-3]Tetracaine is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds with high affinity in the absence of cholinergic agonist (K-eq = 0.5 mu M) and weakly (K-eq = 30 mu M) in the presence of agonist (i.e., to nAChR in the desensitized state). In the absence of agonist, irradiation at 302 nm of nAChR-rich membranes equilibrated with [H-3]tetracaine results in specific photoincorporation of [H-3]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [H-3]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [H-3]tetracaine were subjected to enzymatic digestion, and peptides containing H-3 were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [H-3]tetracaine specifically labeled two sets of homologous hydrophobic residues (alpha Leu(251), beta Leu(257), gamma Leu(260), and delta Leu(265); alpha Val(255) and delta Val(269)) as well as alpha lle(247) and delta Ala(268) within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [H-3]tetracaine-binding site is located within the lumen of the closed ion channel and provides a definition of the surface of the M2 helices facing the channel lumen.