Photoaffinity Labeling the Torpedo Nicotinic Acetylcholine Receptor with [3H]Tetracaine, a Nondesensitizing Noncompetitive Antagonist

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 the Torpedo nicotinic acetylcholine receptor (nAChR). [ 3 H]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 a K 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 for N , N -DEAE and N , 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 of N , N -diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [ 3 H]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 [ 3 H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [ 3 H]tetracaine-binding site. Within α subunit, ≥95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser 173 that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [ 3 H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.