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Separate Subunits for Agonist and Benzodiazepine Binding in the Y- Aminobutyric Acida Receptor Oligomer*

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Separate Subunits for Agonist and Benzodiazepine Binding in the Y- Aminobutyric Acida Receptor Oligomer* T he Journal of Biological Chemistry Vol. 261, No. 32, Issue of November 15, pp. 15013-15016,1986 © 1986 by The American Society of Biological Chemists, Inc. Printed in U.S.A. Separate Subunits for Agonist and Benzodiazepine Binding in the y- Aminobutyric AcidA Receptor Oligomer* (Received for publication, June 24,1986) Stefano O. CasalottiJ, F. Anne Stephenson§, and Eric A. Barnard From the Medical Research Council Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge CB2 2QH, Great Britain The 7-aminobutyric acidA (GABAa) agonist muscimol benzodiazepine-binding site as shown by its photoaffinity can be photoactivated by 254 nm illumination to affin­ labeling by [3H]flunitrazepam applied to the pure receptor ity label its binding site in the GABAa receptor. We protein (1,4). These results have now been confirmed in other have conducted this reaction on the pure receptor from laboratories (Ref. 5 and reviewed in Ref. 6). bovine cerebral cortex in detergent solution, showing Recently, two groups have employed the G ABA agonist, that [3H]muscimol can produce then a specific saturable muscimol, as another photoaffinity reagent to label a GABA- labeling. In the detergent solution, the receptor alone binding site in membranes from rat cerebellum (7, 8). We is sensitive to 254 nm irradiation; this reduces the have adapted this method to label the pure GABA a receptor efficiency of incorporation to below that in the mem­ from bovine cortex and have hence identified the high-affinity branes, but the competing photoreaction with [3H]mus- cimol is sufficient and occurs at a representative set of GABA-binding subunit of the receptor. the muscimol-binding sites, such that it can be em­ MATERIALS AND METHODS ployed for the photolabeling of those sites. The affinity of [3H] muscimol displayed in this irreversible reaction [iV-met/iy(-3H]Flunitrazepam (85 Ci/mmol) and [methylene-3H] is indistinguishable from that of its reversible binding. muscimol (9 Ci/mmol) were from Amersham Int. Flunitrazepam and Ro 7-1986/1 were kindly supplied by Dr. H. Mohler, Hoffmann-La 7-Aminobutyric acid and bicuculline compete in the Roche (Basel, Switzerland), and chlorazepate was a gift from Boeh- photolabeling reaction according to their known affin­ ringer Ingelheim (Bracknell, United Kingdom). Reduced Triton X- ities at the 7-aminobutyric acid-binding site. 100 was from Aldrich, and all other materials and methods not The labeling is shown to occur at the /9-subunit (ap­ specified were as given previously (1, 2). parent Mr 57,000) in the pure receptor. The binding Membrane Receptor Preparations—Bovine brain was dissected and sites for 7-aminobutyric acid agonists, on the /9-sub­ frozen at —80 °C within a maximum of 1 h after removal. Thoroughly units, and the benzodiazepine binding sites, on the a- washed twice frozen-thawed brain membranes were prepared (9), and subunits, are linked allosterically so that a strongly membrane-bound receptor activity was determined using [3H]musci- cooperative hetero-oligomeric structure of this recep­ mol for the measurement of GABA binding in a centrifugation assay tor is deduced. or using [3H] flunitrazepam for the measurement of benzodiazepine binding in a filtration assay (1). Purification and Analysis of the GABAA-Benzodiazepine Receptor— The receptor was purified from bovine cerebral cortex by Ro 7-1986/ 1-agarose affinity chromatography with 0.2% Triton X-100 in the Earlier studies in this laboratory have resulted in the puri­ final media. For some experiments, a reduced form of Triton X-100 fication to homogeneity of the G A BA a* receptor complex of was used in the equilibration and elution of the ion-exchange chro­ bovine (1, 2) or rat (3) brain cortex in media containing matography column. Soluble receptor activity was measured by the polyethylene glycol precipitation-filtration assay (1). Triton X-100, with co-retention of the specific binding sites Samples prepared for SDS-PAGE were first concentrated either for G ABA agonists (recognized by [3H] muscimol) and antag­ by precipitation with 12% (w/v) trichloroacetic acid, washing twice onists and for the brain-type benzodiazepines and for /3- with acetone, and air drying (1) or by precipitation and washing with carbolines. Furthermore, if the media throughout are instead MeOH/CHCb (3:1) (v/v) (10). The samples were dissolved in sample prepared in CHAPS/phospholipid solution, the same complex buffer containing 1 % SDS, denatured instantly at 100 ®C for 5 min is obtained with retention also of the active sites for the in the presence of 10 m M dithiothreitol, and subjected to SDS-PAGE under reducing conditions in 10% (w/v) polyacrylamide slab gels, all chloride ion channel-regulatory ligands and of the allosteric as described by Sigel and Barnard (2). interactions between all of the afore-mentioned sites (2). In For samples which were photoaffinity-labeled, following SDS- both types of preparation, SDS-polyacrylamide gel electro­ PAGE either (i) the slab gels were sliced at 1.5-mm intervals and phoresis revealed only two types of subunit, a (apparent M r extracted and counted as described in Ref. 1 or (ii) the proteins were 53,000) and /9 (apparent M r 57,000). The a-subunit carries the transferred to nitrocellulose by Western blotting as described in Ref. 11. The nitrocellulose was impregnated for 15 min at room tempera­ * The costs of publication of this article were defrayed in part by ture with Autofluor (National Diagnostics, Aylesbury, United King­ the payment of page charges. This article must therefore be hereby dom), and the labeled bands were visualized by fluorography on marked “advertisement" in accordance with 18 U.S.C. Section 1734 preflashed Fuji x-ray film. solely to indicate this fact. Photoaffinity Labeling—Thoroughly washed twice frozen-thawed $ Supported by a graduate studentship from Fidia Laboratories. membranes from bovine brain cortex were diluted to 0.6 mg of § Holder of a Royal Society University research fellowship. To protein/ml with 10 m M HEPES (pH 7.4), 1 m M EDTA, 1 m M whom correspondence should be addressed: MRC Centre, Hills Rd., benzamidine HC1, 1 mg/ml soybean trypsin inhibitor, 1 mg/ml ovo­ Cambridge CB2 2QH, Great Britain. mucoid trypsin inhibitor, 1.25 m M dithiothreitol, 0.02% NaN3. Ali­ 1 The abbreviations used are: GABA, 7 -aminobutyric acid; CHAPS quots (2.35 ml) were incubated for 20 min on ice with [ 3H]muscimol 3 -[(3 -cholamidopropyl)dimethylammonio]-l-propanesulfonate; (60 n M final concentration). Similarly, the purified receptor (0.3 ml HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS- containing 2 pmol of [3H] flunitrazepam-binding sites) was incubated PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. with either [3H] flunitrazepam or [3H] muscimol (each at 100 n M final 15013 15014 Subunits Binding GABA and Benzodiazepines concentration) for 30 min on ice with continuous shaking. These pM muscimol) was much lower in the pure protein than in the samples were irradiated (in 8 -cm diameter flat-bottom vials) with membranes, averaging only 4% of the total irreversible bind­ ultraviolet light from a Camag Universal ultraviolet lamp (peak ing, after 30 min of reaction on ice. wavelength 254 nm) at a distance of 6 cm, again on ice with continuous shaking. The irradiation was interrupted for 2 min at 10-min intervals For the pure receptor alone in Triton X-100 solution, the to avoid overheating of the samples. illumination at 254 nm caused a time-dependent loss of the At the end of the stated total incubation time, the irradiated agonist sites, with the loss of 78% of the initial reversible samples were incubated on ice for 90 min with excess nonradioactive agonist-binding sites with 30 min of illumination. The sites ligand, 10 pM flunitrazepam, or 10 pM muscimol, respectively. For remaining (after 30 min of illumination) then showed the the membrane preparation, the unbound 3H-labeled ligand was re­ same relative susceptibility to the [3H] muscimol photolabel­ moved from the samples by centrifugation at 8000 X g followed by four washes with 10 m M HEPES (pH 7.4) where the first wash ing as the original population. When [3H]muscimol (100 nM) contained also 10 p M muscimol; for the purified receptor, the protein was irradiated for 30 min, it lost 75% of its capacity to bind was precipitated with 12% trichloroacetic acid and washed as de­ reversibly to the pure receptor; on the rat membranes, an 80% scribed above. Control experiments in the dark showed that these loss of its photolabeling capacity upon 10 min of irradiation treatments were sufficient to remove all of the free 3H-labeled ligand. of [3H]muscimol was reported (8). Due to these two competing The acetone-washed pellets were either dissolved for counting of radioactivity with the scintillant described in Ref. 1 or dissolved in reactions, as well as the intrinsically low efficiency of irrever­ sample buffer and subjected to SDS-PAGE under reducing conditions sible attachment of the acylnitrene that has been supposed to as above. form on the photolysis of muscimol (8), the photolabeling of the pure receptor reached only 7.5 ± 0.8% of the sites present RESULTS AND DISCUSSION which can bind [3H] muscimol reversibly. The U V absorption When bovine brain cortex membranes were incubated with of the 0.1% Triton X-100 used as the solvent was also consid­ [3H]muscimol and subsequently irradiated with U V light, it ered as a possible contributory factor to this low efficiency, was found, in agreement with the results of Cavalla and Neff but this detergent could be replaced by a reduced, non-ab­ (8) on rat cerebellar membranes, that tritium was irreversibly sorbing form of Triton X-100 (see “Materials and Methods”) incorporated into the membrane fraction.
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