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J. Biol. Chem., Vol. 277, Issue 51, 50036-50045, December 20, 2002

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Identification of the Bovine -Aminobutyric Acid Type A Receptor  Subunit Residues Photolabeled by the Imidazobenzodiazepine [³H]Ro15-4513^*

Gregory W. Sawyer^§¶, David C. Chiara^¶, Richard W. Olsen^**, and Jonathan B. Cohen

From the  Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California 90095 and the  Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, September 10, 2002, and in revised form, October 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ligands binding to the benzodiazepine-binding site in -aminobutyric acid type A (GABA_A) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABA_A receptor  and  subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [³H]flunitrazepam identified a primary site of incorporation at ₁His-102, whereas studies with [³H]Ro15-4513 suggested incorporation into the ₁ subunit at unidentified amino acids C-terminal to ₁His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified (~200-fold) GABA_A receptor from detergent extracts of bovine cortex, photolabeled it with [³H]Ro15-4513, and identified ³H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of ³H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different  subunits. Based upon this radiochemical sequence analysis, [³H]Ro15-4513 was found to selectively label the homologous tyrosines ₁Tyr-210, ₂Tyr-209, and ₃Tyr-234, in GABA_A receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Aminobutyric acid type A (GABA_A)¹ receptors, the major inhibitory ligand-gated ion channels in the mammalian central nervous system, comprise a family in the Cys loop receptor superfamily of homologous ligand-gated ion channels that also includes glycine and invertebrate glutamate_Cl receptors with anion-selective channels and nicotinic acetylcholine (ACh) and serotonin 5-HT₃ receptors with cation-selective channels (1, 2). Although multiple types of GABA_A receptor exist in vivo differing in subunit composition, distribution, and pharmacological characteristics, each receptor is composed of homologous subunits arranged in a pentamer around a central axis that is the chloride-selective channel. Whereas the most abundant GABA_A receptor in mammalian brain is made up of ₁, ₂, and ₂ subunits in a stoichiometry of 2:2:1 (3, 4), other GABA_A receptors containing two  subunits_(1-6), two  subunits_(1-4), and one additional subunit (, _(1-3), _(1-3), , , and  subunits) can form functional channels (5). Site-directed mutagenesis studies have shown that the two agonist-binding sites in the GABA_A receptor are located extracellularly at - subunit interfaces in a ligand-binding pocket whose residues are partially conserved between superfamily members (6, 7).

Interactions of -aminobutyric acid (GABA) with GABA_A receptors are modulated by many drugs including benzodiazepines, barbiturates, anesthetics, alcohols, and neurosteroids (8, 9). Benzodiazepines are of significant therapeutic interest and are among the most widely prescribed drugs in the world, used to treat anxiety and insomnia, to induce anesthesia, and to reduce seizure activity. The binding of many clinically useful benzodiazepines, such as flunitrazepam, is positively coupled to the agonist-binding sites and results in an allosteric potentiation of apparent GABA binding affinity as measured electrophysiologically. Other drugs, such as the imidazobenzodiazepine Ro15-1788 (flumazenil), can bind to the benzodiazepine site without energetic coupling to the GABA site (zero modulator or benzodiazepine antagonist), whereas for Ro15-4513, an azide analog of Ro15-1788 and the focus of this study, there is negative allosteric coupling with GABA binding (negative modulator or benzodiazepine inverse agonist) for some GABA_A receptor subunit compositions (5, 8-11).

Benzodiazepines bind to a distinct binding site on the GABA_A receptor located between an  and the  subunits. The benzodiazepine-binding site is homologous to the GABA-binding sites located at the - interfaces, with many binding site residues conserved (12, 13). Mutational analyses have identified amino acids in three discrete regions of the primary structure of an  subunit (14) and three regions of a  subunit (15-17) that function as determinants of benzodiazepine binding affinity and/or allosteric coupling to the agonist-binding site. Mutational analyses allow the identification of amino acids that are important determinants of the energetics of ligand binding, but these amino acids need not be direct contributors to the ligand-binding site.

Photoaffinity labeling provides an alternative experimental approach to identify amino acids in proximity to a bound ligand (reviewed in Refs. 18 and 19). For the nicotinic ACh receptor, photoreactive agonists and antagonists have provided extensive identification of amino acids contributing to the agonist-binding sites and to the ion channel (see Refs. 20 and 21 and references therein). For the GABA_A receptor, irradiation at 254 nm results in the covalent incorporation of the agonist [³H]muscimol, with Phe-65 in the bovine ₁ subunit identified as a labeled amino acid (22). The benzodiazepine [³H]flunitrazepam photoincorporates into several  subunit isoforms (23-25), and ₁His-102 has been identified as the primary site of photoincorporation within the ₁ subunit (26, 27). [³H]Ro15-4513 can also be photoincorporated into  subunits of the GABA_A receptor (10), and its site(s) of incorporation was shown to be C-terminal to ₁His-102 (28) and possibly within the transmembrane domains of the receptor (29). Ro15-4513 contains a photoreactive azide that upon UV excitation forms a reactive nitrene. Therefore [³H]Ro15-4513 should photolabel amino acids in close proximity to the azide, and identification of those residues will provide a first definition of the orientation of Ro15-4513 within the benzodiazepine-binding site.

In this report we identify the amino acids photolabeled by [³H]Ro15-4513 in GABA_A receptors purified by affinity chromatography from detergent extracts of bovine cerebral cortex. Labeled amino acids in the ₁, ₂, and ₃ subunits were determined by use of a protein sequencing strategy that depended upon the pattern of ³H release rather than the direct identification of GABA_A receptor subunit amino acids.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [³H]Muscimol (20 Ci/mmol) and [³H]Ro15-4513 (23 Ci/mmol) were from PerkinElmer Life Sciences. Macro-Prep High Q ion exchange resin and chemiluminescent detection reagents were from Bio-Rad. Ro7-1986/1 and flurazepam were the generous gifts of Hoffmann-La Roche, as was monoclonal antibody Bd24 that recognizes selectively the GABA_A receptor ₁ subunit (30). Affinity-purified rabbit antipeptide antibodies specific for the GABA_A receptor ₂, ₃, and ₅ subunits were described previously (31). Triton X-100, reduced Triton X-100, and 6-aminohexanoic acid-Sepharose 4B were from Sigma. Staphylococcus aureus glutamyl endopeptidase (V8 protease) was from ICN Biomedicals. Endoproteinase Lys-C (EndoLys-C) and endoproteinase Asp-N (EndoAsp-N) were from Roche Molecular Biochemicals. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was obtained from Worthington.

Preparation of a Benzodiazepine Affinity Column-- An Ro7-1986/1 benzodiazepine column was prepared using a method similar to Stauber et al. (32) except that the column was prepared by coupling Ro7-1986/1 to 6-aminohexanoic acid-Sepharose 4B rather than to Affi-Gel 202. The amount of coupled Ro7-1986/1 was ~0.45 µmol/ml. The gel was packed into a 2.5-cm outer diameter × 20-cm column and then washed with 1 liter of 50 mM Tris (pH 8.0), 1 M NaCl, followed by 50 mM HEPES (pH 4.5), 1 M NaCl.

Affinity Purification of GABA_A Receptor from Detergent Extracts of Bovine Cortex-- Fresh whole bovine brain was placed on ice, and the meninges were rapidly removed, and the cortex was dissected away from the remaining structures. The cortex was washed to remove blood and stored in 0.32 M sucrose at 80 °C. In preparation for affinity purification of GABA_A receptors, ~100 g of bovine cortex were thawed and homogenized at 4 °C in 10 volumes of 50 mM Tris (pH 8.0) containing 0.32 M sucrose, using a 55-ml Potter-Elvehjem glass homogenizer and Teflon pestle. The homogenate was centrifuged (550 × g, 10 min) to remove particulate matter, and then the pellet, after centrifugation (50,000 × g, 1 h), was resuspended in 20 volumes of distilled water using a Tekmar Ultra-Turrax (Cincinnati, OH). The suspension was centrifuged (50,000 × g, 1 h), and the pellet was resuspended in 10 volumes ice-cold membrane buffer (50 mM Tris, 50 mM KCl, 1 mM EDTA, 10 µg/ml soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 2 mM benzamidine hydrochloride, 0.1 mM benzethonium chloride, 100 µg/ml bacitracin (pH 8.0)). Following another centrifugation (50,000 × g, 1 h), the membrane pellet was stored at 20 °C overnight.

The frozen membrane pellet was thawed on ice and resuspended in 4 volumes of ice-cold solubilization buffer (membrane buffer with 1 M KCl, 0.5% Triton X-100, and 20% glycerol) using an Ultra-Turrax. This suspension was gently stirred for 1 h (4 °C) and then centrifuged (150,000 × g, 1 h). The supernatant was applied three times to an Ro7-1986/1 affinity column (4 °C) pre-equilibrated with 100 ml of solubilization buffer. After loading, the column was washed with 100 ml of solubilization buffer, 400 ml of wash buffer (membrane buffer with 0.2% Triton X-100 and 10% glycerol), and 200 ml of urea wash buffer (wash buffer with 3 M urea). GABA_A receptor was eluted in 5 ml of elution buffer (wash buffer with 3 mM flurazepam and 4 M urea, 1 h of incubation), which was repeated 2 times. Three additional elutions were performed with 30-min incubations (4.5 h total). Following the final incubation, an additional 20 ml of elution buffer was added, and the eluate was pooled (approximately 50 ml) and dialyzed (3 h, 4 °C) against 20 mM Tris (pH 8.0), 0.2% Triton X-100, 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM -mercaptoethanol.

The dialyzed affinity column eluate was applied to a 5-ml High Q ion exchange resin (prepared according to the manufacturer's instructions) and washed extensively (20 mM Tris, 10% glycerol, 0.2% Triton X-100 (pH 7.4)) to remove flurazepam. The protein eluted from the column with 10 ml of 20 mM Tris, 1 M NaCl, 10% glycerol, 0.2% reduced Triton X-100 (pH 7.4) was collected in 1-ml fractions and assayed for [³H]muscimol binding (see under "Binding Assays"). The one or two fractions containing [³H]muscimol binding were pooled and used for photolabeling.

Photoaffinity Labeling of Affinity-purified GABA_A Receptor-- Affinity-purified GABA_A receptors (~100-300 pmol in 5 ml of 50 mM Tris, 10% glycerol, 0.2% reduced Triton X-100 (pH 7.4)) were incubated at 4 °C with 20 nM [³H]Ro15-4513 for 90 min in a glass Petri dish. In some experiments, 10 µM flunitrazepam was included to determine the specificity of [³H]Ro15-4513 photoincorporation. The receptor preparation was irradiated at a distance of 7.5 cm with a 366-nm lamp (UVGL-25 (UVP, Inc., San Gabriel, CA)) for 30 min. After photolabeling, the amount of irreversible binding was estimated by taking 3 aliquots (100 µl) of the photolabeled receptor preparation and incubating them with 1 mM flunitrazepam for 1 h at 4 °C with gentle shaking. Total protein was precipitated, and the retained ³H was determined by filtration as described under "Binding Assays."

Binding Assays-- GABA_A receptor binding was assayed at each step of affinity purification by a [³H]muscimol binding assay (32). Briefly, aliquots of membranes or detergent extracts (100 µl; 0.2-0.006 mg of protein) taken at different steps during affinity purification were incubated in a final volume of 0.5 ml of 50 mM Tris/HCl (pH 7.4) and 50 mM KCl at 4 °C for 30 min with the GABA_A receptor agonist [³H]muscimol at a concentration (40 nM) sufficient to occupy all agonist-binding sites. For solubilized GABA_A receptor, bovine -globulin (100 µl; 30 mg/ml) and 30% polyethylene glycol (300 µl) were added after incubation to precipitate total protein, followed by vigorous vortexing. Receptor-bound [³H]muscimol was trapped by filtration on glass fiber filters (Whatman GF/B). The filters were rinsed with three 2-ml aliquots of ice-cold buffer (50 mM Tris/HCl (pH 7.4), 50 mM KCl, and 7% polyethylene glycol), and retained ³H was determined by liquid scintillation counting. All assays were carried out in triplicate, and nonspecific binding was determined by carrying out incubations in the presence of 0.1 mM GABA in parallel tubes. Protein was estimated using the BCA protein assay (Pierce).

SDS-PAGE and Immunoblotting-- [³H]Ro15-4513-labeled GABA_A receptor was precipitated by methanol and chloroform (33), dissolved in SDS-PAGE sample buffer, and boiled for 5 min, and then the polypeptides were separated by SDS-PAGE (34). Polypeptides were separated on 0.75- or 1.5-mm slab gels consisting of a 10% separating gel and a 3% stacking gel. To visualize separated polypeptides, gels were stained with Coomassie Brilliant Blue. To visualize ³H distribution by fluorography, the gels were soaked in Amplify (Amersham Biosciences) according to manufacturer's instructions and dried prior to exposure to film (X-Omat AR, Eastman Kodak) for 21-28 days at 20 °C.

Tricine gels (16.5% T, 6% C) were used as described (35). To isolate the labeled peptides by SDS-PAGE, the slab gels were cut into 6-mm strips that were eluted for 3 days in 12 ml of 100 mM NH₄HCO₃, pH 8.4, with 0.1% SDS and 2.5 mM dithiothreitol. The eluates were assayed for ³H, and the samples of interest were filtered, concentrated to ~200 µl in Ultrafree Biomax 5K concentrators (Millipore), and precipitated with acetone (75%, 20 °C overnight).

The subunit composition of affinity-purified GABA_A receptors was determined by immunoblot on polyvinylidene difluoride (0.45-µm pore size) membranes after SDS-PAGE using antibodies against the GABA_A receptor ₁ subunit (Bd24, 1:250), ₂ subunit (1:5,000), ₃ subunit (1:1,000), and ₅ subunit (1:1,000). The polypeptides recognized by the primary antibodies were visualized by horseradish peroxidase-conjugated anti-rabbit or mouse IgG (1:5,000) and chemiluminescence.

Enzymatic Digestions-- For digestion with EndoLys-C, samples were resuspended with vortexing in 100-200 µl of 25 mM Tris buffer (pH 8.6) with 0.1% SDS and 500 µM EDTA (EKC buffer). Digestions were performed with 0.3 units of EndoLys-C at 25 °C for 2 weeks. Subsequent digestions were carried out in the same buffer with the following modifications. For EndoAsp-N (0.2 µg, 1 week, 25 °C), the sample was diluted 10-fold with H₂O. For trypsin (10 µg, 3 days, 25 °C), Genapol C-100 (Calbiochem) was added to the sample at 5 times the SDS concentration. No buffer modifications were necessary for V8 protease (50 µg, 3 days, 25 °C). For sequential digestions, the activity of the previous enzyme was inhibited irreversibly by addition of 2 mM diisopropyl fluorophosphate (Sigma) at 25 °C for >2 h.

HPLC-- HPLC fractionation was performed on an Agilent 1100 binary HPLC system with an in-line degasser, column heater, and a diode array absorbance detector. Separations were carried out at 40 °C on a 100 × 2.1-mm Aquapore BU-300 7-µm column (PerkinElmer Life Sciences). Solvent A was 0.08% trifluoroacetic acid in water, and solvent B was 60% acetonitrile, 40% isopropyl alcohol, 0.05% trifluoroacetic acid. The gradient (% solvent B) is indicated in the figures. To neutralize the acid, HPLC fractions were collected in 1.5-ml tubes containing 20 µl of 250 mM Tris (pH 8.1). For enzymatic digestion after HPLC, fractions of interest were rotary-evaporated to near dryness and resuspended as above.

Sequence Analysis-- N-terminal sequence analysis was performed on an Applied Biosystems Procise 492 protein sequencer on Biobrene-treated micro-trifluoroacetic acid filters (ABI 401111). Depending upon the application, two different injection loops were used on the amino acid analyzer. For high sensitivity detection of ³H release, five-sixths of each cycle of Edman degradation were collected for scintillation counting, whereas one-sixth was utilized for residue identification. Although no GABA_A receptor sequences were identified, the other peptide sequences detected were quantified to ensure proper sequencer function. For high sensitivity detection of phenylthiohydantoin-derivatives, two-thirds of each cycle of Edman degradation were used for amino acid analysis, and only one-third was collected for ³H determination. HPLC fractions of interest were drip-loaded directly onto the filters (45 °C) using a syringe pump.

Data Base Searches-- Edman degradation was used to determine the ³H release profile for ³H-labeled subunit fragments that were subjected to sequential proteolytic fragmentation with a panel of side chain-specific proteases. The observed ³H release patterns identified the distribution of Lys, Arg, Asp, and Glu on the N-terminal side of the ³H-labeled amino acid. The experimentally determined distributions of amino acids were used to search protein sequence data bases using the program Pattern Search at pir.georgetown.edu/pirwww/search/patmatch.html. Pattern Search searches for exact matches to proposed sequences in which multiple amino acids can be designated for each position in the sequence. Blast was used to identify the parent proteins of the amino acids sequences identified by Edman degradation (www.ncbi.nlm.nih.gov/BLAST/).

Molecular Modeling-- A model of the extracellular region of the bovine GABA_A receptor (2_1(13-223), 2_{1(10-218) 2(25-233)}) was constructed from the snail acetylcholine-binding protein (AChBP) structure (36) using the Homology module of Insight II on a Silicon Graphic work station. The sequences for the GABA_A receptor subunits were from the National Center for Biotechnology Information (NCB accession numbers P08219, P08220, and P22300), and the coordinates for the AChBP structure (Protein Data Bank code 1I9B) were from the Research Collaboratory for Structural Bioinformatics. Insertions (₁ residues 82, 103, 122-123, 151, and 174-176; ₁ residues 78, 102, 119-120, 144, and 171; and ₂ residues 93, 115, 134-135, 159, and 186) and deletions (in ₁ two residues between 205 and 206; in ₁ two residues between 200 and 201; and in ₂ two residues between 215 and 216) required to facilitate the alignment between the AChBP and the bovine GABA_A receptor subunits were placed at turns to preserve the overall -sheet structure. The ₂ subunit was placed next to an ₁ subunit such that the residues identified previously at the benzodiazepine site were in proximity. Once the pentamer was constructed, the structure was minimized using the Discovery module.

The structures of flunitrazepam and Ro15-4513 were constructed and minimized using the Builder module. Although we were able to build and minimize Ro15-4513, the force fields required for the Docking module are not programmed to accept an azide moiety. This moiety was altered to C=C=O to allow for docking. Structures were placed within the predicted benzodiazepine-binding site located at the interface between the ₁ and ₂ subunits, and the Docking module was used to identify the best ligand orientation. The binding site was defined as ₁ residues 102, 103, 156, 160, 161, 203, 205, 206, 207, 210, and 212 and ₂ residues 54, 58, 77, 79, 81, 98, 130, 132, 138, 140, 142, 144, 184, 185, and 194. For the Ro15-4513 ligand, >200 orientations were sampled, and the minimized best fit (illustrated in Fig. 10) was obtained 117 times. Other orientations of the planar ligand were also sampled, although their interaction energies were not as favorable as the best fit structure. A similar docking was performed with the flunitrazepam model resulting in a prominent single preferred orientation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of GABA_A Receptors-- As summarized in Table I, GABA_A receptor was purified ~100-fold with a 90% recovery from a Triton X-100 extract of membranes from 100 g of bovine cortex on an affinity column of the benzodiazepine Ro7-1986/1 and then concentrated to a small volume by use of an anion exchange column. Ro7-1986/1 is an analog that binds to diazepam-sensitive GABA_A receptors (37). Recombinant GABA_A receptors having a high affinity for diazepam contain the ₂ subunit in combination with  subunits and either ₁, ₂, ₃, or ₅ subunits (for review see Ref. 8). Recombinant GABA_A receptors containing only ₄ or ₆ subunits do not bind diazepam and should not be retained on the Ro7-1986/1 column.

The initial binding capacity (B_max) of the cortical membranes, as assayed by the binding of a saturating concentration of [³H]muscimol, was ~0.6 pmol/mg protein (~1 nmol total). Approximately 80% of the GABA_A receptor was recovered in the Triton X-100 extract, and ~90% of the solubilized GABA_A receptor was retained on the affinity column after washing with 3 M urea. After elution of the column with 3 mM flurazepam in 4 M urea, ~90% of the solubilized GABA_A receptor (730 pmol) was recovered in 50 ml with a [³H]muscimol binding capacity 200-fold higher than the initial cortical membranes. The eluate was dialyzed to remove urea and to reduce the flurazepam concentration, and anion exchange chromatography was then used to further remove fluazepam and to concentrate the receptor. This step did effectively concentrate the GABA_A receptor into a volume of 1-2 ml, although recovery was poor (20-50%), and there was no further purification.

Subunit Composition of Affinity-purified GABA_A Receptor-- In previous investigations, GABA_A receptors affinity-purified with an Ro7-1986/1 affinity column from detergent extracts of bovine or rat cortex were found to contain ₁, ₂, and ₃ subunits (31, 38). To identify the GABA_A receptor  subunits present in the affinity column eluate after High Q ion exchange chromatography, immunoblot analysis was performed using antibodies specific for the ₁, ₂, ₃, or ₅ subunit. An antibody specific for the ₁ subunit recognized a 51-kDa band, the anti-₂ subunit antibody recognized a 50-kDa band, and the anti-₃ subunit antibody recognized a 56-kDa band as well as a 29-kDa band, presumably an ₃ subunit proteolytic fragment (Fig. 1). Anti-₅ subunit antibodies did not recognize any polypeptides in the eluate from the Ro7-1986/1 column (data not shown).

View larger version (85K): [in this window] [in a new window]   Fig. 1.   Identification of  subunits contained in GABAA receptors purified from bovine brain. GABAA receptors were purified from Triton X-100 extracts of bovine cortex using a Ro7-1986 affinity column and concentrated on an anion exchange column as described under "Experimental Procedures." Aliquots (5 µg of protein/lane) of the purified receptor were fractionated by analytical SDS-PAGE (10% acrylamide). After electrophoretic transfer, the membrane was cut into strips containing individual lanes that were incubated with anti-1 (Bd24; 1:250), anti-2 (1:500), or anti-3 (1:1,000) GABAA receptor subunit antibodies, and secondary antibody for detection by chemiluminescence. The mobilities of prestained protein standards are indicated on the left.

Photolabeling Affinity-purified GABA_A Receptor with [³H]Ro15-4513-- Affinity-purified GABA_A receptor (40 nM muscimol sites) was equilibrated with 20 nM [³H]Ro15-4513 and then photolabeled. After photolabeling, ~50% of the [³H]Ro15-4513 appeared irreversibly incorporated, as judged by the amount remaining bound 1 h after the addition of 1 mM flunitrazepam as competitor. When GABA_A receptors were photolabeled in the absence or presence of 10 µM flunitrazepam and the ³H-labeled polypeptides determined by SDS-PAGE, an ~50-kDa band was the primary site of flunitrazepam-inhibitable labeling, along with additional specific ³H incorporation in several bands between 46 and 57 kDa (Fig. 2). The mobilities of the labeled bands were generally consistent with the protein bands recognized by antibodies against bovine GABA_A receptor ₃, ₁, and ₂ subunits (Fig. 1).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Affinity-purified bovine GABAA receptor photolabeled with [3H]Ro15-4513 demonstrates benzodiazepine site specificity. A, preparations of affinity-purified GABAA receptor (~250 pmol [3H]muscimol-binding sites in 5 ml) were photolabeled with [3H]Ro15-4513 (20 nM) in the absence (F) and presence (+F) of flunitrazepam (10 µM). An aliquot (200 µl) of each photolabeling reaction was precipitated by methanol/chloroform, and the recovered protein was separated by SDS-PAGE (10% acrylamide). Shown is the 3H distribution on the resulting gel as detected by fluorography. The mobilities of protein standards are depicted on the left. B, chemical structures of Ro15-4513 and flunitrazepam.

Radiosequence Analyses Using Sequential Proteolytic Fragmentation-- Based upon the binding of [³H]muscimol to the affinity-purified GABA_A receptor preparation, the receptors were purified ~200-fold from the crude membrane preparation, and active GABA_A receptor comprised ~2% of the protein in the preparation. In addition, the receptor preparation was heterogeneous, with some receptors containing ₁ subunits and others with ₂ or ₃ subunits. We reasoned that for this heterogeneous, partially purified preparation, it might be difficult to purify by HPLC and/or SDS-PAGE a labeled subunit fragment to a sufficient extent to allow direct identification of the labeled peptide by the released phenylthiohydantoin-derivatives (lower detection limit of ~0.5 pmol). However, the high radiochemical specific activity of [³H]Ro15-4513 (23 Ci/mmol) enabled us to develop a radiosequence strategy to identify the labeled amino acid in the presence of non-labeled contaminating fragments. Whereas 1 pmol of [³H]Ro15-4513-labeled protein would contain ~18,000 cpm, sequence analysis of samples containing as little as 2,000-3,000 cpm could lead to a clear identification of the cycle of Edman degradation containing the labeled amino acid, even though the level of labeled peptide would be too low for amino acid detection.

To identify the sites of photoincorporation by [³H]Ro15-4513 in GABA_A receptor subunits, we used a panel of proteases with defined side chain specificities to cleave the labeled subunit fragments and Edman sequencing with ³H detection in each cycle (radiosequence analysis) to determine the number of amino acids between the site of cleavage and the labeled residue(s). For example, after digestion with EndoLys-C, which cleaves at the C-terminal side of lysines, the release of ³H after n cycles of Edman degradation would indicate that ³H was incorporated in a peptide containing a lysine n amino acids before the labeled residue. For this analysis, we utilized EndoLys-C, trypsin (cleavage C-terminal of lysines and arginines), V8 protease (cleavage C-terminal of glutamates), and EndoAsp-N (cleavage N-terminal of aspartates). By use of sequential digestions with as many a four enzymes, we could identify the distribution of these amino acids (Lys, Arg, Asp, and Glu) on the N-terminal side of the site(s) of labeling, and we hoped that this empirically determined distribution would produce a unique identification of the site(s) of [³H]Ro15-4513 incorporation.

Isolation of Material for Radiosequence Analysis-- Affinity-purified GABA_A receptor (200 pmol) was photolabeled with [³H]Ro15-4513 and separated by preparative slab gel SDS-PAGE (10% acrylamide). The resulting gel was cut into 6-mm strips, and each strip was eluted and counted for ³H as shown in Fig. 3A. A broad peak of ³H was detected spanning three bands (6-8) centered at 56 kDa, and ~80% of the ³H in these bands was recovered after concentration, precipitation, and resuspension. This region corresponded to the gel region containing the ₁, ₂, and ₃ subunits identified by immunoblot (Fig. 1). Because the ₃-immunoreactive material had lower mobility (higher M_r) than the ₁ or ₂ bands, we analyzed the sites of ³H incorporation separately for Bands 6 and 8 with the hope that each would contain different GABA_A receptor  subunits.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Purification by HPLC of [3H]Ro15-4513-labeled GABAA receptor subunit fragments. An affinity-purified preparation of bovine GABAA receptor (~250 pmol in 5 ml) was equilibrated with [3H]Ro15-4513 (20 nM) and irradiated as described under "Experimental Procedures." The labeled material was separated by preparative SDS-PAGE (10% acrylamide), and the gel was then cut into 6-mm strips. A, total 3H distribution recovered from the eluted gel bands as determined by liquid scintillation counting of 1% aliquots. A broad peak of 3H was centered at Band 7 (~56 kDa), and an additional peak was seen at the dye front. When the eluates of the bands were concentrated, precipitated, and resuspended in preparation for proteolysis, >77% of the 3H was recovered in Bands 6-8, whereas <7% of the 3H in the dye front was recovered. The eluates from Bands 6-8 were digested individually with EndoLys-C, and the resulting fragments were separated by reversed-phase HPLC as described under "Experimental Procedures." The 3H distributions () are shown as well as the absorption at 214 nm () and the % solvent B (- - -) for Band 6 (B), Band 7 (C), and Band 8 (D). For Band 6 (~66 kDa), 80,000 cpm were injected; total recovery was 80%, and ~50% of 3H recovered a single peak (fractions 22 and 23). For Band 7 (~56 kDa), 72,000 cpm were injected, total recovery was 70%, with ~40% in fractions 22 and 23. For Band 8 (~48 kDa), 90% of the 114,000 cpm injected was recovered, with ~40% in fractions 22 and 23.

Identification of [³H]Ro15-4513 Incorporation at ₃Tyr-234-- The sequential digestion strategy that we used to characterize the site(s) of [³H]Ro15-4513 photoincorporation within the GABA_A receptor subunit(s) isolated from Band 6 (centered at 66 kDa) is summarized in Fig. 4A. When an aliquot of Band 6 (2,000 cpm) was sequenced, no release of ³H (<2 cpm above background) was detected in 25 cycles of Edman degradation (data not shown). Material from Band 6 was digested with EndoLys-C, and when the digest was fractionated by reversed-phase HPLC (Fig. 3B), there was a peak of ³H in fractions 22-23 (~43% organic) which was pooled for further analysis. Radio-sequence analysis of an aliquot of this pool (5,500 cpm, Fig. 5A) revealed no prominent peak of ³H release, although there was a gradual increase in the background ³H release during the 30 cycles of Edman degradation that would be consistent with gradual wash off of the labeled peptide from the filter. The remainder of the pool was digested with EndoAsp-N. When an aliquot of that digest was sequenced (5,500 cpm, Fig. 5B), there were peaks of ³H release in cycles 19 (76 cpm) and 27 (34 cpm). When the remaining material was digested with V8 protease and an aliquot sequenced (5,500 cpm, Fig. 5C), there was a peak of ³H release in cycle 9 (324 cpm) with no evidence of the ³H release seen previously in cycles 19 and 27. The remainder of the material was digested with trypsin. When this digest was sequenced (5,500 cpm, Fig. 5D), there was ³H release in cycle 6 (476 cpm) with no ³H release remaining in cycle 9. 

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Digestion strategy for analysis of [3H]Ro15-4513-labeled GABAA receptor subunit fragments.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   3H release upon sequencing successive digestions of material from Band 6; identification of 3 Tyr-234 as a site of [3H]Ro15-4513 photolabeling. Fractions 22 and 23 (28,000 cpm) from the HPLC separation of the EndoLys-C digest of Band 6 (Fig. 3B) were pooled, rotary-evaporated, and resuspended in 100 µl of EKC buffer (22,000 cpm recovered). A, an aliquot from the EndoLys-C digest was sequenced. B, the remaining material was digested with EndoAsp-N from which an aliquot was sequenced. C, the remaining material was then digested with V8 protease, and an aliquot of that digest was sequenced. D, the remainder was digested with trypsin and then sequenced. The sequential digests were performed as described under "Experimental Procedures," and for each sample equal amounts of 3H (5500 cpm) were loaded on the filter and sequenced for 30 cycles of Edman degradation, after which 1160 (A), 780 (B), 410 (C), and 300 (D) cpm remained on the filter. After the initial EndoLys-C digest (A), no release of 3H above background was detected other than a 50 cpm anomaly in cycle 23 which was not reproduced in two other sequencing studies of similarly prepared samples. Upon digestion with EndoAsp-N, release of 3H was seen in cycles 19 and 27 (B). 3H release in those cycles was eliminated by digestion with V8 protease, which resulted in 3H release in cycle 9 (C). After digestion with trypsin, 3H release was shifted to cycle 6 (D). The observed patterns of 3H release following sequential digestion with this panel of proteases were consistent with only one stretch of bovine GABAA receptor subunit primary sequence and identifies 3Tyr-234 as a site of [3H]Ro15-4513 photoincorporation (E).

This observed pattern of ³H releases after sequential proteolysis indicated the following. 1) One or more residues labeled by [³H]Ro15-4513 occurred 18 and/or 26 amino acids after an Asp residue. 2) This same labeled residue(s) occurs 9 amino acids after a Glu (they are the same site(s) because the ³H release seen after EndoAsp-N treatment was no longer present after digestion with V8 protease). 3) The same labeled residue(s) occurs 6 amino acids after an Arg (not a Lys, because EndoLys-C did not initially cut there). A search of the bovine GABA_A receptor subunit sequences determined that this pattern of amino acids (DX₇DX₈EX₂R) occurred only in the ₃ subunit, with sites of cleavage at Asp-208, Asp-216, Glu-225, and Arg-228 identifying ₃Tyr-234 as the amino acid labeled by [³H]Ro15-4513 (Fig. 5E). The ³H release seen in cycles 19 and 27 after EndoAsp-N digestion would be accounted for by [³H]Ro15-4513 incorporation at ₃Tyr-234 and partial cleavage at either Asp-216 or Asp-208. The only inconsistency with this conclusion is the fact that V8 protease produced no cleavage at ₃Glu-233, the amino acid preceding ₃Tyr-234. However, a plausible explanation for the lack of cleavage at ₃Glu-233 is that the modification at ₃Tyr-234 prevents V8 protease action at the peptide bond between ₃Glu-233 and the modified tyrosine.

To determine the uniqueness of this pattern of amino acid residues, we used the program Pattern Search to search the PIR data base for the pattern 3(X_K)-D-7(X_KD)-D-8(X_KD)-E-2(X_KDE)-R-4(X_KDER)-2X where X_K = not K; X_KD = not K or D; X_KDE = not K, D, or E; and X_KDER = not K, D, E, or R. From this search we identified 43 sequences that matched this distribution, only six of which were mammalian. Four of the mammalian sequences were the ₃ subunit of the GABA_A receptor (from different species); the other 2 sequences were variants of human intestinal mucin 2. The only bovine sequence identified was the GABA_A receptor ₃ subunit.

Identification of [³H]Ro15-4513 Incorporation at ₁Tyr-210-- We next characterized the site(s) of [³H]Ro15-4513 photoincorporation within the GABA_A receptor subunit(s) isolated from Band 8 (centered at 48 kDa, Fig. 3A). A summary of the digestion strategy is presented in Fig. 4B. Sequence analysis of an aliquot (2,000 cpm) of Band 8 revealed no ³H release (<2 cpm above background) in 25 cycles of Edman degradation (data not shown). When the Band 8 material was digested with EndoLys-C and fractionated by reversed-phase HPLC, the ³H elution profile (Fig. 3D) was similar to that seen for Band 6, with a peak of ³H in fractions 22 and 23 (~45% organic). When these fractions were pooled and an aliquot was sequenced (8,500 cpm, Fig. 6A), there was no release of ³H in 30 cycles of Edman degradation other than a gradual increase in background radioactivity. When the remainder of the pool was digested with trypsin and an aliquot was sequenced (8,500 cpm, Fig. 6B), there was a peak of ³H release in cycle 23 (148 cpm). When the rest of the material was digested with EndoAsp-N and an aliquot was sequenced (8,500 cpm, Fig. 6C), there was a prominent peak of ³H in cycle 12 (397 cpm) and no evidence of the ³H release previously seen in cycle 23. 

View larger version (27K): [in this window] [in a new window]   Fig. 6.   3H release upon sequencing successive digestions of material from Band 8; identification of 1 Tyr-210 as a site of [3H]Ro15-4513 photolabeling. Fractions 22 and 23 (40,000 cpm) from the HPLC separation of the EndoLys-C digestion of gel Band 8 (Fig. 3D) were pooled, rotary-evaporated, and resuspended in 200 µl of EKC buffer (38,000 cpm recovered). A, an aliquot from the EndoLys-C digest was sequenced. B, the remaining material was digested with trypsin from which an aliquot was sequenced. C, the remaining material was then digested with EndoAsp-N, and an aliquot of that digest was sequenced. The sequential digests were performed as described under "Experimental Procedures," and for each sample equal amounts of 3H (8600 cpm) were loaded on the filter and sequenced for 30 cycles of Edman degradation, after which 1550 (A), 620 (B), and 450 (C) cpm remained on the filter. No release of 3H above background was seen when the initial EndoLys-C digest was sequenced (A). After digestion with trypsin, prominent release of 3H was seen in cycle 23 (B). 3H release in cycle 23 was eliminated by digestion with EndoAsp-N, which resulted in a new release of 3H in cycle 12 (C). The observed patterns of 3H release following sequential digestion with this panel of proteases were consistent with only one stretch of bovine GABAA receptor subunit primary sequence and identify 1Tyr-210 as a site of [3H]Ro15-4513 photoincorporation (D).

This pattern of ³H releases after sequential proteolysis indicated the following. 1) A residue labeled by [³H]Ro15-4513 occurred 23 amino acids after an Arg residue (not a Lys, because EndoLys-C did not cleave at that position). 2) This same labeled residue occurs 11 amino acids after an Asp. 3) There were no lysines within 30 amino acids of the labeled amino acid. A search of the amino acid sequences for the bovine GABA_A receptor subunits found that this pattern of specific residues occurs only in the ₁ subunit. Cleavage at Arg-187 and Asp-199 would identify ₁Tyr-210 as the amino acid labeled by [³H]Ro15-4513 (Fig. 6D). Significantly, ₁Tyr-210 is homologous to ₃Tyr-234.

We also analyzed the occurrence of this pattern with the PIR Pattern Search program (6(X_K)-R-11(X_KR)-D-9(X_KRD)-2X where X_K = not K; X_KR = not K or R; and X_KRD = not K, R, or D). This pattern was far from unique; 10,823 protein sequences were identified of which 41 were of published bovine proteins. However, the ₁ subunit was the only GABA_A receptor subunit sequence to match this pattern.

Evidence of [³H]Ro15-4513 Incorporation at ₂Tyr-209-- Material from Band 7 (~56 kDa) was also digested with EndoLys-C and fractionated by reversed-phase HPLC (Fig. 3C). When aliquots from fractions 20-22 were sequenced individually, similar ³H release patterns were seen for fractions 20 and 21 that differed from the release seen for fraction 22 (Fig. 7). For fractions 20 and 21, when samples containing 800 and 1550 cpm were sequenced, there were peaks of ³H release in cycle 6 of 95 and 176 cpm, respectively. In contrast, radio-sequence analysis of a sample of fraction 22 containing 1730 cpm had only 17 cpm released in cycle 6. Sequence analysis of fractions 20-21 from the HPLC of Bands 6 and 8 also revealed 100-200 cpm ³H release in cycle 6 (data not shown), which had not been seen in the sequencing of the peak ³H fractions (Figs. 5A and 6A). An examination of the amino acid sequence of the bovine GABA_A receptor ₂ subunit in the region homologous to the identified sites of [³H]Ro15-4513 photolabeling in the ₁ and ₃ subunits indicated that ³H release in cycle 6 was consistent with labeling of the ₂ subunit at Tyr-209, which is homologous to ₁Tyr-210 and ₃Tyr-234. EndoLys-C digestion of the ₂ subunit should produce a 16-amino acid fragment containing Tyr-209 in cycle 6 (Fig. 7B). This peptide is shorter than the labeled peptides produced by EndoLys-C digestion of the ₁ or ₃ subunits (64 and 43 amino acids, respectively), and the shorter peptide would be expected to elute at a lower % organic solvent. Because the first digestion was with EndoLys-C, there were no other proteases that would cleave between ₂Lys-203 and ₂Tyr-209. Comparison of the ₂ and ₃ subunit sequences in this region reveals that the only difference in protease sites is an Arg-Lys substitution 6 amino acids before the labeled tyrosines. Thus, without the initial digestion with EndoLys-C, the ³H release pattern seen for labeled ₂ subunit material after digestion with EndoAsp-N, V8 protease, or trypsin would be the same as we observed for the labeled ₃ subunit (Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   3H release upon sequencing HPLC fractions 20 and 21 from EndoLys-C digests consistent with [3H]Ro15-4513 photolabeling at 2Tyr-209. A, 3H release profiles during 30 cycles of Edman degradation for the shoulder of 3H (fractions 20 and 21) from the HPLC purification of an EndoLys-C digest of material from Band 7 (Fig. 3C) are shown (, fraction 20, 800 cpm loaded, 30 cpm remaining; , fraction 21, 1,550 loaded, 75 cpm remaining). Also shown are the 3H release patterns seen when the major peak of 3H from that HPLC purification was sequenced (, fraction 22, 1,730 loaded, 270 remaining), as well as the Band 7 material prior to EndoLys-C digestion (, 2,000 cpm loaded, 490 remaining after 25 cycles). B, release of 3H occurred in cycle 6 for both fractions 20 and 21, which was consistent with cleavage after 2Lys-203 and [3H]Ro15-4513 photoincorporation at 2Tyr-209, a residue that is homologous to both 1Tyr-210 and 3Tyr-234.

Bovine Mitochondrial F₁-ATPase Peptides Co-purify with ³H-Labeled GABA_A Receptor Fragments-- Based upon the identified sites of [³H]Ro15-4513 incorporation in GABA_A receptor  subunits, EndoLys-C digestion of labeled ₁, ₂, and ₃ subunits would generate labeled fragments of 64, 16, and 43 amino acids. In preliminary experiments on an analytical scale, we established that digestion with EndoLys-C produced an ~7-kDa fragment that by radiosequence analysis after redigestion with EndoAsp-N had a ³H release profile consistent with the presence of the ₁ subunit fragment labeled at ₁Tyr-210. We attempted to purify the ³H-labeled ₁ subunit fragment from an EndoLys-C digest by a combination of Tricine SDS-PAGE and reversed-phase HPLC. When the labeled subunits were separated by SDS-PAGE, the peak of ³H migrated at ~56 kDa (Fig. 8A, band 10), and this incorporation was reduced by >85% when photolabeling was carried out in the presence of 10 µM flunitrazepam. When material eluted from Band 10 was digested with EndoLys-C and the digest fractionated by Tricine SDS-PAGE, there were specifically labeled bands of 16, 8, and 4 kDa (Fig. 8B) (and similar ³H distributions for the EndoLys-C digests of Bands 9 and 11). When the 8-kDa band was excised, eluted, and purified by reversed-phase HPLC (Fig. 8C), the majority of the ³H was recovered in a single peak at 47% organic (fraction 23). When this fraction was sequenced, no bovine GABA_A receptor subunit sequences were detected, but there was a clear primary sequence identified in 30 cycles of Edman degradation that was a fragment of the bovine mitochondrial F₁-ATPase A chain (1E1QA) beginning at Thr-3 (initial yield, 3 pmol). There were also three or four other peptides present at initial levels of 1-2 pmol, although no clear sequence assignment was possible. Based upon the radiochemical specific activity of [³H]Ro15-4513, the 6,500 cpm sequenced was incorporated in ~0.4 pmol of labeled GABA_A receptor subunit fragment (and ~1 pmol of unlabeled subunit fragment if the labeled and unlabeled peptides remained together throughout the purification.) This level of GABA_A receptor subunit fragment would not have not been detectable in the presence of a complex mixture of peptides at the ~2-3 pmol level. Fractions 24 and 25 were also sequenced and were found to contain fragments from the F₁-ATPase chain D (1E79D) beginning at Leu-406 and Ala-9, respectively, at initial yields of ~0.5 pmol and identified in 20 cycles of Edman degradation. Once again, no GABA_A receptor subunit sequences were detected.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   SDS-PAGE/HPLC purification of a [3H]Ro15-4513-labeled GABAA receptor fragment was insufficient for sequence detection via Edman degradation. A, affinity-purified GABAA receptor (~50 nM [3H]muscimol sites) was photolabeled with [3H]Ro15-4513 (20 nM) in the absence (filled bars) and presence (open bars) of flunitrazepam (10 µM). After photolabeling, polypeptides were fractionated by preparative SDS-PAGE (10% acrylamide), and the gels were cut into 6-mm slices and eluted. The total 3H recovered from each of the gel slices is shown along with the mobilities of the molecular weight standards. B, material recovered from Band 10 (~56 kDa (128,000 cpm flunitrazepam, 16,000 cpm +flunitrazepam)) was digested with EndoLys-C, and the digest was fractionated by Tricine SDS-PAGE with the distribution of 3H determined after elution from the gel slices. C, the material recovered from Band J (14,700 cpm flunitrazepam, 400 cpm +flunitrazepam) was purified by reversed-phase HPLC. The 3H distribution (, flunitrazepam, 10% of each fraction counted) is shown as well as the absorption at 214 nm () and the % solvent B (- - -). No bovine GABAA receptor subunit peptide sequences were detected when the peak of 3H or adjacent fractions were sequenced, although other bovine sequences were identified (see text).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The goal of this study was to use photoaffinity labeling to identify the amino acid(s) in the benzodiazepine-binding site that is photolabeled by [³H]Ro15-4513, an imidazobenzodiazepine that contains an azide substituent that upon UV illumination will produce a reactive nitrene. Because of the defined structure of the photoreactive intermediate, identification of the amino acids labeled by [³H]Ro15-4513 will define the orientation of the ligand within the benzodiazepine-binding site. Previous studies have shown that for receptors containing the ₁, ₂, ₃, or ₅ subunits, the binding of Ro15-4513 was negatively coupled to the binding of GABA, in contrast to the positive allosteric coupling seen for flunitrazepam at those receptor subtypes or for Ro15-4513 binding to receptors containing ₄ or ₆ subunits (11).

[³H]Flunitrazepam has been shown to photoincorporate into ₁His-102 (26, 27), which had been identified by mutational analyses as a major affinity determinant of benzodiazepine binding (39). ₁His-102 is also an important determinant of Ro15-4513 efficacy, as substitutions at that position regulate equilibrium binding affinity (40) and determine whether it acts as a positive or negative modulator of GABA responses (41). However, the extent of overlap between the binding site for flunitrazepam and Ro15-4513 is uncertain. Photolabeling of recombinant ₁₃₂ GABA_A receptors by Ro15-4513 resulted in a 90% decrease of high affinity [³H]flunitrazepam or [³H]Ro15-4513-binding sites, but after photolabeling by flunitrazepam there was a 90% reduction of flunitrazepam sites, whereas the number of [³H]Ro15-4513 sites were reduced by less than 10% (42). Although [³H]flunitrazepam is photoincorporated into ₁His-102, the amino acid(s) photolabeled by [³H]Ro15-4513 are contained within a subunit fragment extending from residue 104 to the C terminus of the ₁ subunit (43), possibly within amino acids 247-289 that span the end of the first transmembrane segment to the beginning of the third transmembrane segment (29).

In this report we have used a radiochemical sequencing strategy to identify the amino acids labeled by [³H]Ro15-4513 in a heterogeneous preparation of GABA_A receptors purified from bovine cortex on a benzodiazepine affinity column. The purification procedure resulted in an ~200-fold purification of a population of receptors containing, based upon immunoblot analysis, ₁, ₂, or ₃ subunits. Based upon the level of specific [³H]muscimol binding, active GABA receptors comprise ~1% of the protein in the preparation, and, not surprisingly, GABA_A receptor subunits or subunit fragments could not be directly detected by Edman degradation when labeled subunits were isolated by SDS-PAGE or when subunit fragments were further purified by SDS-PAGE and reversed-phase HPLC. However, by use of N-terminal sequence analysis to determine the patterns of ³H release as labeled subunit fragments were digested sequentially with a panel of four proteases, we established that [³H]Ro15-4513 is photoincorporated into homologous positions in the three subunits: ₁Tyr-210, ₂Tyr-209, and ₃Tyr-234. Our results are most compelling for the ₃ subunit, for which positive identification of the ³H release profile was made after digestion with 4 proteases, whereas the identification of the labeled amino acids in the ₁ and ₂ subunits utilized only 3 and 1 proteases, respectively. Although the subunit heterogeneity of the brain GABA_A receptor preparation initially complicated the identification of the sites by making it appear that multiple sites of labeling might exist within a single subunit, the fact that our data are consistent with photoincorporation into homologous Tyr in all three subunits actually strengthens the conclusion about each individual identification.

The position in the rat GABA_A receptor (₁Tyr-209) occupied by the tyrosine residues photolabeled by [³H]Ro15-4513, as well as ₁Thr-206, have been shown by site-directed mutagenesis to affect the affinity of ligands whose binding is coupled either positively or negatively to the GABA site (14, 44). In addition, ₁Val-211 and the corresponding ₅Ile-215 have been identified as imidazobenzodiazepine affinity determinants (45). In a pharmacophore model of the benzodiazepine-binding site developed based upon the results of mutational analyses (46), ₁Tyr-209 was proposed to interact with all ligands. However, in the model it was not positioned in proximity to the azide of Ro15-4513 (or to ₁His-101).

The radiosequence strategy that we have used permits a positive identification of [³H]Ro15-4513-labeled amino acids in the ₁, ₂, and ₃ subunits, but in the absence of direct identification of subunit masses by Edman degradation, our results provide no information about the relative efficiency of incorporation of [³H]Ro15-4513 into the different subunits or about the relative abundance of each subunit in our receptor preparation. It is most likely that the labeled ₁, ₂, and ₃ subunits are each contained within distinct populations of monomeric GABA_A receptors. However, further immunoaffinity purification by use of subunit-specific antibodies would allow a direct determination of whether labeled ₁ and ₃ subunits, for example, coassemble in a single receptor. For rat cortical membranes, both ₁ and ₃ subunits were photolabeled with [³H]Ro15-4513, and based upon immunoaffinity purification with subunit specific antisera, a minor proportion of labeled ₁ subunits copurifies with ₃ subunits (47).

No Evidence of [³H]Ro15-4513 Labeling of ₁His-102 or Amino Acids within ₁ 97-117-- Previous photolabeling and mutational studies of the GABA_A receptor indicate that at least 2 additional regions of an  subunit primary structure may come in contact with Ro15-4513 at the benzodiazepine-binding site, specifically residues near ₁His-102 and ₁Tyr-160 (7, 13). An examination of an alignment of the bovine _1-3 sequences for these regions (Fig. 9) indicated that if they were labeled, we would have seen patterns of ³H release after proteolytic fragmentation significantly different from those that we observed in our experiments. For the regions preceding ₁His-102 and ₁Tyr-160, all three subunits would produce the same patterns of protease cleavages, EX₁₉KX₄D and EX₄DX₆K, respectively. Although we have no direct proof that cleavages at these sites occurred, the efficiency of cleavages that occurred before ₃Tyr-234 and the corresponding regions of the ₁ and ₂ subunits makes it likely that these cleavages also occur. The patterns of ³H release we observed lead us to conclude that there is no significant [³H]Ro15-4513 photoincorporation in ₁His-102, in the corresponding positions in the ₂ or ₃ subunits, or in nearby amino acids. The ³H release patterns seen during sequence analysis of the major peak of ³H in the HPLC fractionation of EndoLys-C digests of GABA_A receptor subunits (Figs. 5 and 6) are also not consistent with labeling at or near ₁Tyr-160, but the ³H release in the sixth cycle of Edman degradation of fractions 20 and 21 of the EndoLys-C digest (Fig. 7), which we attribute to labeling of ₂Tyr-209, is also consistent with labeling of ₁Tyr-162 or the corresponding position in the other subunits. However, in other experiments when the materials eluted from gel Bands 6-8 were digested with EndoAsp-N and then sequenced, ³H release was limited to cycles 12 and 19 with no release in cycle 14 as would be expected for labeling of ₁Tyr-162 (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 9.   Sequence alignments of GABAA receptor subunit segments containing benzodiazepine-binding site affinity determinants. Shown are alignments of segments of the bovine 1, 2, and 3 subunits along with segments of the 2 subunit with residues (*) identified by mutational analyses as benzodiazepine affinity determinants (7, 16). Predicted cleavage sites for the proteases used in this study are denoted in boldface type. The homologous tyrosines photolabeled by [3H]Ro15-4513 are enclosed in a box. Segments A-E and -sheet 1 are identified by homology to the agonist site primary structure elements in the AChBP and the nicotinic ACh receptor.

A Model of the Benzodiazepine-binding Site-- To gain further appreciation of the selective labeling of ₁Tyr-210 by [³H]Ro15-4513, we constructed an homology model of an extracellular domain of GABA_A receptor containing 2 ₁, 2 ₁, and a ₂ subunits, based upon the crystal structure of the snail AChBP, a soluble, secreted protein homologous to the extracellular domain of the nicotinic ACh receptor (36, 48). Shown in Fig. 10 is a stereo representation of the benzodiazepine-binding site within our model with Ro15-4513 docked in an energetically favored orientation. In this orientation the photoreactive nitrogen of the azide is ~5 Å from ₁Tyr-210 and 4 Å from ₁His-102, the residue that is labeled by [³H] flunitrazepam, and it is also within 4-6 Å of ₁Tyr-160, ₁Ser-205, and ₂Phe-77. In a docking simulation with flunitrazepam (not shown), we found that it was oriented with its nitro group within 4 Å of ₁His-102, consistent with one proposed mechanism of photoincorporation via a nucleophilic attack on a benzene carbon ortho to the nitrogen (49), and within 5 Å of ₁Tyr-210.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Stereo representation of Ro15-4513 bound within a GABAA receptor benzodiazepine-binding site. An homology model of the extracellular region of the bovine GABAA receptor (112) was constructed from the crystal structure of the molluscan AChBP (36), and Ro15-4513 was docked in the benzodiazepine-binding site located at the 1-2 subunit interface. The -sheets from the AChBP structure are denoted (1, 2,  ... ). Ro15-4513 is in stick representation with atom types denoted: carbon, black; nitrogen, blue; and oxygen, red (compare with Fig. 2B). Hydrogens were excluded for simplification. GABAA receptor amino acids previously identified by mutational analyses as potential contributors to the benzodiazepine-binding pocket are denoted in ball and stick representation. These residues are contained within 3 primary structure segments from both the 1 and 2 subunits that are each indicated by different colors. The 1 residues localized to the benzodiazepine site are 1His-102 (yellow); Tyr-160, Tyr-162, and Thr-163 (red); and Gly-201, Ser-205, Thr-207, Tyr-210, and Val-212 (blue); the 2 residues are Met-57 and Tyr-58 (brown); Phe-77, Gly-79, and Thr-81 (green); and Met-130 and Thr-142 (magenta). In this orientation the photoreactive nitrogen of Ro15-4513 (*) was ~5 Å from 1Tyr-210 (cyan) and 4 Å from 1His-102.

In terms of the model, the labeling of ₁Tyr-210 by [³H]Ro15-4513 and the lack of labeling of ₁His-102 is very striking. This might result from the preferential reaction of the photoreactive intermediate with Tyr rather than His, as photolabeling of tyrosines by azide photoaffinity labeling has been frequently observed (19), whereas labeling of His has not been reported to our knowledge. However, the labeling of ₁Tyr-210 also occurs in the absence of labeling of ₁Tyr-160. Alternatively, the lack of labeling of ₁His-102 or ₁Tyr-160 may, in fact, occur because the structure of the benzodiazepine-binding site occupied by Ro15-4513 may differ significantly from that in the homology model. Because the binding of flunitrazepam is coupled positively to the binding of GABA, whereas the binding of Ro15-4513 is coupled negatively, flunitrazepam will stabilize the same conformation of the GABA_A receptor that is favored by the binding of GABA, whereas Ro15-4513 will stabilize a conformation with low affinity for GABA, presumably the resting state of the receptor. Changes of the accessibility of substituted cysteines for chemical modification have provided evidence that the binding of GABA results in a change in the structure of the benzodiazepine-binding site (50).

The homology model is based upon the structure of the AChBP in a single conformational state (36), one that binds ACh with high affinity and is presumed to be closest in structure to a nicotinic ACh receptor conformation that binds ACh with high affinity, i.e. either an open channel or desensitized state. A recent comparison of the structure of the AChBP with that of the extracellular domain of the Torpedo nicotinic ACh receptor in the resting state identifies significant differences in structure within the agonist-binding site (51). In future studies it will be important to determine whether [³H]Ro15-4513 photolabels amino acids other than ₁Tyr-210 when photolabeling is carried out in the presence of GABA or another agonist.

	FOOTNOTES

^* This work was supported in part by United States Public Health Service Grant GM 58448 and by an award to the Harvard Medical School from the Howard Hughes Medical Institute Biomedical Research Support Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by National Institutes of Health Training Grant F32NS11015. Present address: Dept. of Pharmacology and Physiology, College of Osteopathic Medicine, Oklahoma State University, Tulsa, OK 74107.

^¶ Both authors contributed equally to this work.

^** To whom correspondence may be addressed: Dept. of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095. Tel.: 310-825-5093; E-mail: rolsen@mednet.ucla.edu.

To whom correspondence may be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen@hms.harvard.edu.

Published, JBC Papers in Press, October 17, 2002, DOI 10.1074/jbc.M209281200

	ABBREVIATIONS

The abbreviations used are: GABA_A receptor, GABA receptor type A; GABA, -aminobutyric acid; ACh, acetylcholine; AChBP, molluscan acetylcholine-binding protein; V8 protease, S. aureus glutamyl endopeptidase; EndoLys-C, endoproteinase Lys-C; EndoAsp-N, endoproteinase Asp-N; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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