The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.     

A significant part of native gamma-aminobutyric AcidA receptors containing alpha4 subunits do not contain gamma or delta subunits.

Using a novel antibody directed against the alpha4 subunit of gamma-aminobutyric acidA (GABAA) receptors, 5% of all [3H]muscimol but only about 2% of all [3H]Ro15-4513 binding sites present in brain membrane extracts could be precipitated. This indicated that part of the alpha4 receptors containing [3H]muscimol binding sites did not contain [3H]Ro15-4513 binding sites. Immunoaffinity purification and Western blot analysis of alpha4 receptors demonstrated that not only alpha1, alpha2, alpha3, beta1, beta2, and beta3 subunits but also gamma1, gamma2, gamma3, and delta subunits can be colocalized with alpha4 subunits in native GABAA receptors. Quantification experiments, however, indicated that only 7, 33, 4, or 7% of all alpha4 receptors contained gamma1, gamma2, gamma3, or delta subunits, respectively. These data not only explain the low percentage of [3H]Ro15-4513 binding sites precipitated by the anti-alpha4 antibody but also indicate that approximately 50% of the alpha4 receptors did not contain gamma1, gamma2, gamma3, or delta subunits. These receptors, thus, either are composed of alpha4 and beta1-3 subunits only, or additionally contain epsilon, pi, or so far unidentified GABAA receptor subunits.     

Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety

To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.     

Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.     

Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.     

Preparation of Antibodies to β Subunits of γ-Aminobutyric AcidA Receptors

Antisera were produced in rabbits against synthetic peptides based on two regions of the cDNA sequence of the beta 1 subunit of bovine gamma-aminobutyric acidA (GABAA) receptors. The deduced amino acid sequences were similar in other beta subunits of bovine, rat, and chick receptors, predicting cross-reactability with all beta subunits. One antiserum (anti-beta e) was raised against an extracellular moiety near the invariant disulfide loop thought to be located near the neurotransmitter binding domain; the other (anti-beta c) was raised against an intracellular moiety containing a consensus sequence for cyclic AMP-dependent protein kinase phosphorylation of a serine residue. Predicted secondary structures suggested high potential immunogenicity for the chosen antigen peptides. Both antisera at high dilutions recognized the same polypeptide bands on western blots of GABAA receptors purified from three regions of bovine brain (four bands at 57, 54, 53, and 52 kDa in cerebral cortex) but fewer bands (57, 54, and 52 kDa) in hippocampus and cerebellum (one major band at 54 kDa, traces at 57 and 53 kDa). This is consistent with the presence of multiple beta subunits whose expression varies with brain region, as shown by molecular cloning. The anti-beta c antibody was able to immunoprecipitate purified GABAA receptor [3H]-muscimol binding, 87% in bovine cortex and 75% in total rat brain; the anti-beta e was unable to immunoprecipitate any antigen. These antibodies indicate a region-dependent heterogeneity of beta subunits and should be useful for analyzing structure, function, and localization of GABAA receptor subtypes in brain.     

Differential Responses of Expressed Recombinant Human γ-Aminobutyric AcidA Receptors to Neurosteroids

Neuroactive steroids, in particular 3 alpha-hydroxypregnanes, are allosteric modulators of the gamma-aminobutyric acidA (GABAA) receptor. Regionally selective expression of receptor subunit subtypes may account for differential responsiveness of tissues to GABAergic inhibition and neurosteroid modulatory effects. The effect of 5 alpha-pregnan-3 alpha-ol-20-one (epiallopregnanolone) on heterotropic cooperativity on the GABAA receptor complex has been studied in three subtypes of expressed recombinant human receptors and in rat brain and spinal cord. Steroid potentiation of [3H]flunitrazepam binding was greatest for the alpha 3 beta 1 gamma 2 receptor complex, whereas alpha 1 beta 1 gamma 2 and alpha 2 beta 1 gamma 2 complexes showed less than 100% enhancement in binding. Previous studies suggest that the spinal cord is devoid of alpha 1, whereas cerebellum is rich in alpha 1 subunits. Correspondingly, a differential enhancement of [3H]flunitrazepam binding in spinal cord (51%) versus cerebellum (28%) was also observed. The structure of neuroactive steroids is important in determinikng the extent of neuromodulatory activity. The 5 beta-pregnanes,5 beta-pregnan-3 alpha-ol-20-one (epipregnanolone) and 5 beta-pregnan-3 alpha,21-diol-20-one (5 beta-tetrahydrodeoxycorticosterone), were both less potent than their corresponding 5 alpha derivatives. A 3 alpha hydroxyl group is essential for neuromodulatory activity in the expressed receptors, as demonstrated by the observation that 5 alpha-pregnan-3 beta-ol-20-one (allopregnanolone) and 4-pregnen-3, 20-dione (progesterone) were both inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.     

Chronic ethanol consumption enhances internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex

The molecular mechanisms that underlie ethanol dependence involve alterations in the functional properties and subunit expression of GABAA receptors. Chronic ethanol exposure decreases GABAA receptor alpha1 subunits and increases alpha4 subunit levels in cerebral cortical membranes. This study explored the effect of chronic ethanol exposure on internalization of GABAA/benzodiazepine receptors. Chronic ethanol exposure increased alpha1 subunit levels by 46 +/- 12% and [3H]flunitrazepam binding by 35 +/- 9% in the clathrin-coated vesicle (CCV) fraction. There was a corresponding 34 +/- 8% decrease in alpha1 peptide expression and 37 +/- 6% decrease in [3H]flunitrazepam binding in the synaptic fraction. Chronic ethanol consumption also increased the alpha1 subunit immunoprecipitate in the cytosolic fraction (77 +/- 22%), measured by western blot analysis. Moreover, co-immunoprecipitation of both clathrin and adaptin-alpha with alpha1 subunits was increased in the cytosolic fraction, suggesting that alpha1 subunit endocytosis is enhanced by chronic ethanol consumption. In contrast, alpha4 subunit peptide levels were not altered in the CCV fraction despite a 39 +/- 13% increase in peptide levels in the synaptic fraction of cortex. Moreover, acute ethanol exposure did not alter alpha1 subunit peptide expression or [3H]flunitrazepam binding in the synaptic or CCV fractions. These results suggest that chronic ethanol consumption selectively increases internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex.     

Immunological Identification of Multiple α-Like Subunits of the γ-Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.     

Ion and Temperature Effects on the Binding of γ-Aminobutyrate to Its Receptors and the High-Affinity Transport System

A set of procedures was developed to study the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na(+)-dependent transport carrier, at 25 and 37 degrees C in the presence of physiological concentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze-thawing or treatment with Triton X-100. Isoguvacine, (-)-baclofen, and (-)-nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high-(KD less than 30 nM), middle- (KD = 100-500 nM), and low-affinity (KD greater than 5 microM) binding sites. However, the binding data in the middle-affinity region (100-1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl- at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl-. Apparent binding to the transport carrier required both Na+ and Cl-. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high-affinity site of the GABAA receptor and the transport binding site.     

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor alpha 4 subunit.

A cDNA of rat brain encoding the GABAA receptor alpha 4 subunit has been cloned. Recombinant receptors composed of alpha 4, beta 2 and gamma 2 subunit bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine 'alcohol antagonist' [3H]Ro 15-4513, but fail to bind benzodiazepine agonists. The alpha 4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The alpha 4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.     

Peptide Subunits of γ-Aminobutyric AcidA/Benzodiazepine Receptors from Bovine Cerebral Cortex

The gamma-aminobutyric acidA/benzodiazepine receptor complexes from bovine cerebral cortex were purified by immunoaffinity chromatography, and the main component peptide subunits were characterized. The peptide band originally thought to be a single beta subunit [57,000 Mr band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] is composed of at least four different peptides of 54,000-57,000 Mr. Two peptides of 55,000 and 57,000 Mr were recognized by the beta subunit-specific monoclonal antibody 62-3G1. Peptides in the range of 54,000-57,000 Mr were photoaffinity-labeled with [3H]muscimol. A different 57,000 Mr peptide was photoaffinity-labeled by [3H]flunitrazepam, but neither was recognized by the monoclonal antibody 62-3G1 nor photoaffinity-labeled with [3H]muscimol. Some peptides could be identified by their differential mobility shift in SDS-PAGE after treatment with endoglycosidase H. Two additional subunit peptides of 51,000 and 53,000 Mr were also photoaffinity-labeled by [3H]flunitrazepam and reacted with antiserum A. However, the 57,000 Mr peptide that also was photoaffinity-labeled by [3H]flunitrazepam did not react with antiserum A.     

Inducibility of protein-reactive antibodies by peptide immunization: comparison of three epitope peptides of hen egg-white lysozyme.

Three epitope peptides of hen egg-white lysozyme (HEL) were tested for ability to induce antibodies reactive with native HEL. Each peptide was coupled to bovine gamma-globulin (B gamma G) and 4 rabbits were immunized with each peptide-B gamma G conjugate in complete Freund's adjuvant. The mean association constants (K0s) of HEL-reactive antibodies (HEL-R-Abs) from each immunizing group to [3H]acetyl HEL or to [3H]acetyl-peptide were measured in solution by a double antibody method. Only peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) induced high-affinity antibodies to HEL (K0 = 2.5 x 10(6)-2.3 x 10(7) M-1) among the three epitope peptides tested. The association constants of antipeptide loop I.II to [3H]acetyl peptide loop I.II were always one to two orders of magnitude higher than those to HEL. In addition, 50 to 80% of the anti-peptide loop I.II antibodies were reactive with native HEL. The specificity of anti-peptide loop I.II was directed to a conformational feature of the peptide rather than to native HEL and reactivity of the antibody to HEL was interpreted as a kind of cross-reaction. The HEL-R-Abs from anti-Ploop I.II antisera also manifested neutralizing activities against the enzymic activity of HEL when Micrococcus luteus was used as the substrate.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium. Contribution of a nucleophilic amino acid residue of the alpha1 subunit.

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.     

Purification of the γ-Aminobutyric AcidA/Benzodiazepine Receptor Complex by Immunoaffinity Chromatography

The bovine gamma-aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62-3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7-1986/1. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the immunoaffinity-purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7-1986/1 affinity-purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS-PAGE.