Biochemistry 1988, 27, 2337-2345
2337
Photoaffinity Labeling of the Acetylcholine Binding Sites on the Nicotinic Receptor by an Aryldiazonium Derivative+ Jocelyne Langenbuch-Cachat,t Cassian Bon,* Christophe Mulle,II Maurice Goeldner,*-t Christian Hirth,*,t and Jean-Pierre Changeux" Laboratoire de Chimie Bioorganique, Laboratoire Associg au CNRS (UA 31). UniversitP Louis Pasteur, FacultP de Pharmacie, 67048 Strasbourg Cgdex. France, UnitP des Venins et Unite AssociPe Institut PasteurIINSERM (UA 285), Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris CZdex 15, France, and Neurobiologie MolPculaire et Laboratoire AssociP au CNRS (UA 041 149), Interactions Molgculaires et Cellulaires, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris CPdex 15, France Received August 27, 1987; Revised Manuscript Received November 10, 1987
ABSTRACT: p-(Dimethy1amino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E . electricus electroplaque to agonists. In vitro, it reduces the a-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T . marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the a subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of D D F at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M . P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-3011. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per a-bungarotoxin-binding site suffices to fully block a-bungarotoxin binding to the membrane-bound receptor. Thus, D D F behaves as a monovalent photoaffinity label of the acetylcholine-binding site.
%e nicotinic acetylcholine receptor (AcChoR)' from fish electric organ and vertebrate neuromuscular junction is a well-characterized membranebound allosteric protein [reviews in Anholt et al. (1984), Changeux et al. (1984), Popot and Changeux (1984), and Hucho (1986)l made up of four polypeptide chains associated into a heterologous pentamer with an accepted stoichiometry of a#$ (Reynolds & Karlin, 1978; Lindstrom et al., 1979; Raftery et al., 1980). The cDNAs coding for all four of the subunits have been cloned and sequenced in several species [reviews in Numa et al., (1983), Changeux et al. (1984), and Stroud and Finer-Moore (1985)l and several models for the transmembrane organization of these subunits inferred from the primary structure data [reviews in Popot and Changeux (1984) and Stroud and FinerMoore (1985)l. The test of these models by the localization of functional domains within the primary structure of the subunits remains one of the more challenging issues in AcChoR research. One approach consists in the use of affinity and photoaffinity ligands to covalently label defined sites (Jacoby & Wilchek, 1977; Chowdry & Westheimer, 1979) followed by the identification and positioning of the labeled This investigation was supported in part by funds from the MinistEre de la Recherche et de I'Enseignement Sufirieur, the Centre National de la Recherche Scientifique, the Fondation pour la Recherche Mtdicale, the Institut National de la Santt et de la Recherche MEdicale, the Colltge de France, the Commissariat 5 I'Energie Atomique, the Fondation Fyssen, and the Muscular Dystrophy Association of America. f Universitt Louis Pasteur. 8 Unitt des Venins Institut Pasteur. 11 Neurobiologie Moltculaire Institut Pasteur.
amino acid(s) along the primary structures of the subunits [see Kao et al. (1984), Giraudat et al. (1986, 1987), and Oberthur et al. (1986)l. The AcChoR protein carries several distinct categories of binding sites. The nicotinic agonists and competitive antagonists bind, at equilibrium, to two primary acetylcholine (AcCho) binding sites that are selectively tagged by snake venom a-toxins [reviews in Karlin (1983) and Changeux et al. (1984)l. The noncompetitive blockers (NCB) of the permeability response bind to several classes of sites, among which is a high-affinity site present as a single copy per receptor that specifically binds histrionicotoxin and phencyclidine (PCP) (Heidmann et al., 1983; Oswald et al., 1983). The first probe used to label the agonist-binding sites was p(trimethylammoniumy1)benzenediazonium fluoroborate (TDF) (Changeux et al., 1967; Mautner & Bartels, 1970; Wieland, et al., 1979). Incorporation of about one molecule of this affinity reagent per a-toxin-binding site was sufficient to completely block AcCho binding to the receptor (Wieland et al., 1979). Several less chemically reactive alkylating analogues of AcCho have also been used with success [reviews in Karlin (1969, 1983)]; however, these sulfhydryl-directed affinity ligands require the prior reduction of at least one I Abbreviations: DDF, p(dimethy1amino)benzenediazonium fluoroborate; TDF, p(trimethylammoniumy1)benzenediazonium fluoroborate; PCP, phencyclidine; AcCho, acetylcholine; AcChoR, acetylcholine receptor; NCB, noncompetitive blocker; NaDodSO,, sodium dodecyl sulfate; DEAE, diethylaminoethyl; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
0006-2960/88/0427-2337$01.50/0 0 1988 American Chemical Society
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BIOCHEMISTRY
disulfide bond on the AcChoR and it is known that this modification alters the functional properties of the AcChoR protein (Karlin, 1969; Chang & Bock, 1977; Barrantes, 1980; Walker et al., 1984). In all of these experiments, the affinity label was incorporated exclusively into the a-subunit [review in Karlin (1983)], suggesting that the two AcCho-binding sites are carried, at least in part, by the two a-chains of the AcChoR molecule. Photoaffinity labels appear more appropriate to study the topology of the AcCho-binding sites since the photogenerated species possess much higher reactivity (Chowdry & Westheimer, 1979; Bayley & Knowles, 1977). The photosensitive derivatives used up to now with the nicotinic AcChoR were analogues to either AcCho (Kiefer et al., 1970; Hucho et al., 1976; Witzeman & Raftery, 1977) or a-bungarotoxin (Witzeman & Raftery, 1978; Hucho, 1979; Witzeman et al., 1979; Nathanson & Hall, 1980), and they all labeled the a-subunit and, to variable extents, the other AcChoR subunits. However, no stoichiometries of irreversible inactivation were presented for these compounds, making difficult the interpretation of these data in terms of authentic photoaffinity labeling. In this paper we study the photoaffinity labeling of the primary AcCho-binding sites on the native, unreduced AcChoR using p-(dimethy1amino)benzenediazonium fluoroborate (DDF). This diazonium ion is fairly stable under physiological conditions and gives rise, after irradiation, to the corresponding aryl cation, which is known for its hyperreactivity (Ambroz & Kemp, 1979, 1982; Himeshima et al., 1985; Grieve et al., 1985; Kieffer et al., 1986). We have previously shown that DDF can be used to label the high-affinity-binding site for NCBs (Kotzyba-Hibert et al., 1985). In the present study we demonstrate that, in the presence of PCP, DDF is an efficient photosensitive irreversible probe of the AcCho-binding sites on the native receptor. The efficiency of the labeling experiments was improved by inducing the photoaffinity labeling via an energy-transfer reaction between a tryptophan of the receptor protein and the photosensitive ligand (Goeldner & Hirth, 1980). Such selective labeling, based on the preferential photodecomposition of the ligand when bound to the active sites, enables us to extend to a receptor protein the concept of “photosuicide inactivation” previously described with acetylcholinesterase (Goeldner et al., 1982). MATERIALS AND METHODS Materials Electrophorus electricus were obtained from Paramount Aquarium (Ardsley, New York), and live Torpedo marmorata were provided by the Institut Universitaire de Biologie Marine (Arcachon, France). [3H]-a-Toxin from Naja nigricollis was a gift from Drs. Morgat, Menez, and Fromageot from the Commissariat 21 1’Energie Atomique (Saclay, France). [ m e t h ~ l - ~ -N,N-Dimethyl-”-( H] butyloxycarbony1)-pphenylenediamine was a gift of Drs. Van Hove and Rousseau. a-Bungarotoxin from Bungarus multicinctus was from Boehringer (Mannheim, FRG). Millipore filters (HAWP 02500, 0.45 pm) were obtained from Millipore (Mutzig, France). DEAE 81 paper circles were from Whatman (England), and d-tubocurarine and carbamoylcholine were from Sigma (St. Louis, MO). All other chemicals were of analytical grade from Prolabo (Paris, France), E. Merck (Darmstadt, FRG), or Fluka (Buchs, Switzerland). Fluorescence spectra were recorded with a Jobin-Yvon (JY 3C) spectrofluorometer using 1 cm x 1 cm quartz cells. Absorbance spectra were obtained with a Jobin-Yvon
L A N G E N B U C H - C A C H A T ET A L .
(Duospac 203) spectrophotometer. For irradiation experiments, monochromatic light was obtained from a 1000-W xenon-mercury lamp (Hanovia) connected to a grating monochromator (Jobin-Yvon, France). The light intensity was measured with a thermopile (Kipp and Zohnen, Netherland) coupled to a microvoltmeter, the response of which is independent of the wavelength of the incident light between 260 and 500 nm. For example, at 350 nm light measurement of 1 mV corresponds to an incident energy of 2.17 X 10” einstein s-l cm-,. An iris diaphragm was introduced in the light beam between the source and the monochromator and adjusted in order to vary the number of incident photons in the assay cuvette. E. electricus physiological solution was 1.5 mM sodium phosphate buffer (pH 7.0), 150 mM NaCl, 5 mM KCl, 2 mM CaCl,, and 2 mM MgCl,. Phosphate buffer for irradiation experiments and for initial velocity measurements of [3H]-a-toxin binding was 10 mM sodium phosphate (pH 7.0) and 150 mM NaCl. The buffer for the determination of [3H]-a-toxin-bindingsites at equilibrium was 10 mM Tris-HC1 (pH 7.0), 10 mM NaCl, and 1% Triton X-100. Methods (I) Synthesis of [ m e t h ~ l - ~ H l D D FThe . starting material was [methyl-3H]-N,N-dimethyl-N’-(butyloxycarbonyl)-pphenylenediamine (specific activity 60 Ci/mmol) synthesized at the Commissariat 21 1’Energie Atomique (Saclay, France). The radioactive precursor (100 mCi in 5 mL of ethanol) was diluted by 29.5 mg (0.125 nmol) of unlabeled material. After cooling to -80 OC, the solvent was removed under vacuum ( mmHg). The remaining solid residue was then dissolved in 0.25 mL of 34% (w/v) fluoroboric acid at room temperature under inert atmosphere and stirred for 10 min. After the solution was cooled at -10 OC, sodium nitrite (8.5 mg, about 10% excess) was added in small amounts over a period of 30 min and stirred for an additional 45 min in the dark. The yellow crystals were filtered off (4.2 mg), and the filtrate was taken up in a minimum of acetone and precipitated with anhydrous ether (7.2 mg). The overall yield was 40% and the specific activity 0.28 Ci/mmol. The purity of the diazonium salt was checked by UV absorbance spectrum with reference to a pure sample. DDF can be kept in aqueous solution M) at -30 OC without noticeable damage for over 1 year. ( 2 ) Electroplaque Experiments. The pharmacological effect of DDF was examined on an isolated electroplaque preparation from E. electricus according to the methods of Schoffeniels and Nachmansohn (1957) and Higman et al. (1963) by recording the membrane potential with a glass microelectrode. ( 3 ) Single-Channel Recordings. AcCho-activated singlechannel currents were recorded in the dark in the “outside-out” mode from myotubes of the mouse cell line C2, which was kindly provided by C. Pinset (Institut Pasteur, Paris). Myotubes were bathed in a solution of the following composition: NaCl, 140 mM; KCl, 4 mM; CaCl,, 1.2 mM; MgCl,, 1 mM; glucose, 11 mM; and Hepes, 5 mM; pH 7.2. Electrodes were filled with an intracellular solution containing 150 mM KCl and a low concentration of Ca2+ (
