TY - JOUR T1 - Photoaffinity Labeling the Torpedo Nicotinic Acetylcholine Receptor with [<sup>3</sup>H]Tetracaine, a Nondesensitizing Noncompetitive Antagonist JF - Molecular Pharmacology JO - Mol Pharmacol SP - 290 LP - 299 DO - 10.1124/mol.56.2.290 VL - 56 IS - 2 AU - Richard E. Middleton AU - Nina P. Strnad AU - Jonathan B. Cohen Y1 - 1999/08/01 UR - http://molpharm.aspetjournals.org/content/56/2/290.abstract N2 - Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in theTorpedo nicotinic acetylcholine receptor (nAChR). [3H]Tetracaine binds at equilibrium to a single site with a K eq value of 0.5 μM in the absence of agonist or presence of α-bungarotoxin and with aK eq value of 30 μM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen forN,N-DEAE andN,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester ofN,N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [3H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR α, β, γ, and δ subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [3H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [3H]tetracaine-binding site. Within α subunit, ≥95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser173 that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [3H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain. ER -