TY - JOUR T1 - Identification of Amino Acids of the Torpedo Nicotinic Acetylcholine Receptor Contributing to the Binding Site for the Noncompetitive Antagonist [<sup>3</sup>H]Tetracaine JF - Molecular Pharmacology JO - Mol Pharmacol SP - 300 LP - 307 DO - 10.1124/mol.56.2.300 VL - 56 IS - 2 AU - Martin J. Gallagher AU - Jonathan B. Cohen Y1 - 1999/08/01 UR - http://molpharm.aspetjournals.org/content/56/2/300.abstract N2 - [3H]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 μM) and weakly (K eq = 30 μM) 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 [3H]tetracaine results in specific photoincorporation of [3H]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [3H]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [3H]tetracaine were subjected to enzymatic digestion, and peptides containing3H were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [3H]tetracaine specifically labeled two sets of homologous hydrophobic residues (αLeu251, βLeu257, γLeu260, and δLeu265; αVal255 and δVal269) as well as αIle247 and δAla268 within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [3H]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. ER -