Photoaffinity Labeling the Torpedo Nicotinic Acetylcholine Receptor with [H]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]Tetracaine binds at equilibrium to a single site with a Keq value of 0.5 mM in the absence of agonist or presence of a-bungarotoxin and with a Keq value of 30 mM 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]tetracaine resulted in covalent incorporation with similar efficiency into nAChR a, b, g, and d subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [H]tetracainebinding site. Within a subunit, $95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain. The muscle nicotinic acetylcholine receptor (nAChR) consists of four homologous subunits (a2bgd) arranged pseudosymmetrically around a central axis that is a cation-selective ion channel. Each subunit has a common primary structure motif: a hydrophilic, extracellular N-terminal half containing amino acids of the agonist-binding sites, followed by three hydrophobic membrane spanning segments (M1–M3), a cytoplasmic domain, a fourth transmembrane segment (M4), and short extracellular C-terminal tail. Affinity labeling studies, site-directed mutagenesis, and low-resolution (9 Å) cryoelectron microscopy provide considerable information about nAChR structure (reviewed in Karlin and Akabas, 1995; Hucho et al., 1996; Unwin, 1998). The two agonistbinding sites, which are located extracellularly at the a-g and a-d subunit interfaces, are composed of multiple loops of primary structure from a and g (or d) subunits (reviewed in Prince and Sine, 1998). M2 domains from each subunit line the pore of the ion channel (Imoto et al., 1988; Unwin, 1995), with additional contributions from the extracellular ends of the M1 segments (Zhang and Karlin, 1997), whereas M3 and M4 segments are more peripheral and in contact with lipid (Blanton and Cohen, 1994). Noncompetitive antagonists (NCAs) block the nAChR permeability response without preventing the binding of ACh (acetylcholine). A structurally diverse group of drugs act as NCAs, including many aromatic amines, general anesthetics, fatty acids, steroids, and neuropeptides such as Substance P (reviewed in Arias, 1998). Studies of the binding of the aromatic amines [H]meproadifen and [H]phencyclidine (PCP) or of the spiropiperidine [H]histrionicotoxin ([H]HTX) to nAChR-rich membranes from Torpedo electric organ establish that each binds with high affinity (K ; mM) to one site per nAChR and to additional lower-affinity sites (Heidmann et al., 1983). The high-affinity site is linked allosterically to the ACh site, with most aromatic amines binding at equilibrium with highest affinity in the presence of agonist [i.e., to the desensitized state of the nAChR (Cohen et al., 1985; Arias, 1996; Lurtz and Pedersen, 1999)]. This work was supported in part by U.S. Public Health Service Grant NS19522. 1 Present address: Merck Research Laboratories, Rahway, New Jersey 07065. ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; NCA, noncompetitive antagonist; V8 protease, Staphylococcus aureus glutamyl endopeptidase; GSSG, oxidized glutathione; [H]HTX, [H]histrionicotoxin; [I]TID, 3-(trifluoromethyl)-3-(M-[I]iodophenyl)diazirine; HTX, dl-perhydrohistrionicotoxin; H10-HTX, dl-decahydro(pentenyl)histrionicotoxin; PAGE, polyacrylamide gel electrophoresis; PCP, phencyclidine; TPS, Torpedo physiological saline. 0026-895X/99/020290-10$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:290–299 (1999). 290 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from Affinity-labeling studies have identified homologous residues near the cytoplasmic end of each M2 segment that contribute to the high-affinity binding site in desensitized Torpedo nAChRs for the aromatic amine NCAs [H]chlorpromazine (Revah et al., 1990) and [H]trimethylphenylphosphonium (Hucho et al., 1986). Mutational analyses also implicate these amino acids as affinity determinants for the aromatic amine QX-222 (Charnet et al., 1990) and for aliphatic alcohols (Forman, 1997) acting as open channel blockers. However, in each conformational state, there may be distinct, nonoverlapping sites for positively charged NCAs, and different regions within the ion channel may contribute to the binding site for a single ligand in different conformational states. In the desensitized nAChR, [H]meproadifen mustard reacts with aGlu at the extracellular end of M2 (Pedersen et al., 1992), and based on fluorescence energy transfer, the binding site for ethidium is located above the level of the bilayer in the vestibule of the channel (Johnson and Nuss, 1994, but see Lurtz et al., 1997). In the open channel state, [H]quinacrine azide is photoincorporated into amino acids within aM1 (DiPaola et al., 1990), and mutations within aM1 affect quinacrine potency as an NCA but not chlorpromazine (Tamamizu et al., 1995). Photoaffinity labeling studies with 3-(trifluoromethyl)-3-(M-[I]iodophenyl) diazirine ([I]TID; White and Cohen, 1992) and [H]diazofluorene (Blanton et al., 1998), uncharged, hydrophobic NCAs, have identified a binding site in the M2 domain in the absence of agonist (closed channel), as well as changes in structure of the M2 domain between resting and desensitized states. However, the TID site in the closed channel appears distinct from the binding site for PCP because PCP does not inhibit [I]TID photoincorporation and TID does not inhibit [H]PCP binding (White et al., 1991). Tetracaine (dimethylaminoethyl-p-butylaminobenzoate) is an unusual aromatic amine NCA because it is 100-fold more potent as an inhibitor of [H]HTX binding (K ' 1 mM) in the absence of agonist than in the presence (Blanchard et al., 1979), and it stabilizes the resting state rather than the desensitized state of the nAChR (Boyd and Cohen, 1984). Here, we characterize the binding properties of several structural analogs of tetracaine to further define the requirements for preferential binding to the closed channel state, and we use [H]tetracaine itself as an intrinsic photoaffinity reagent to define the structure of its high-affinity binding site in the nAChR in the absence of agonist. [H]Tetracaine is specifically photoincorporated with similar efficiency into each nAChR subunit. In the following report (Gallagher and Cohen, 1999), we identify the homologous amino acids in the M2 segment of each subunit that contribute to this high-affinity [H]tetracaine site. Experimental Procedures Materials. [ring-3,5-H]Tetracaine ([H]tetracaine, 36 Ci/mmol) and [H]HTX (60 Ci/mmol) were prepared at New England Nuclear Research Products (Boston, MA) by tritium gas catalytic reduction of 3,5-dibromotetracaine and dl-decahydro(pentenyl)histrionicotoxin (H10-HTX), respectively. For binding experiments, [ H]tetracaine was purified to .95% by silica thin-layer chromatography (5:4:1 cyclohexane/chloroform/diethylamine; Rf 5 0.17). When stored in ethanol, [H]tetracaine decomposed at ;10% per month, forming tritiated degradation products that did not partition into nAChRrich membranes or bind to glass filters. Also, these degradation products did not appear to photoincorporate into nAChR-rich membranes because the same [H]tetracaine photolabeling patterns were seen with [H]tetracaine of 95 or 50% radiochemical purity (not