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). [H-3]Tetracaine binds at equilibrium to a single site with a K-eq value of 0.5 mu M in the absence of agonist or presence of alpha-bungarotoxin and with a K-eq value of 30 mu M 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 [H-3]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [H-3]tetracaine concentration establish that the observed photolabeling is at the high-affinity [H-3]tetracaine-binding site. Within a subunit, greater than or equal to 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 [H-3]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.