Abstract

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.