Antibodies Recognising the GABA_A/Benzodiazepine Receptor Including Its Regulatory Sites

Abstract

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.

The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

“…Lane G is taken from a separate immunoblot, hence two markers are shown. (Stephenson et al, 1986a was clearly revealed by the antibody reaction under comparable conditions ( Figure Ic, track F).…”

“…Thus, deglycosylation of the purified receptor was carried out using endoglycosidase F treatment and the products were detected by immunoblotting. After this treatment, it was found with an a subunit-specific polyclonal antibody (Stephenson et al, 1986a) that the a subunit disappeared and a major product about 8000 lower in Mr than the original a (Mr in the range 44-46 000, in three experiments) was detected in parallel with a minor product about 5000 lower in Mr than a (Mr in the range 48-49 000) (Figure 3a). When the final products from this treatment of the intact, denatured receptor were revealed by antibody 1A6, these, plus two additional products of Mr 57 000 (minor) and 55 000 derived from the ,3 subunit were found ( Figure 3B).…”

“…In this preparation, the purified receptor was shown to have a high affinity specific binding site for GABA agonists and antagonists, a high affinity specific binding site for the benzodiazepine agonists, antagonists and inverse agonists, and also a low affinity GABA binding site detected by the GABA agonist facilitation of benzodiazepine binding. The receptor preparation was homogeneous in several separation systems, one of which was narrow-range isoelectric focusing, where a single band of pI 5.6 was found (Stephenson et al, 1986a). SDS-polyacrylamide gels of the isolated GABAA receptor carried out under reducing conditions and stained with either Coomassie Blue or silver showed the presence of only the a and (3 subunits (Figure la,b).…”

Identification of the α3‐subunit in the GABAA receptor purified from bovine brain

“…Flunitrazepam and Ro 7-1986/1 were gifts from Dr H. Mohler (Hoffman-La Roche, Basel, Switzerland) and chlorazepate was a gift from Boehringer lngelheim (Bracknell, England). All other materials were as noted elsewhere [3,17].…”

“…GABA and benzodiazepine receptor-binding assays, and SDS-PAGE under reducing conditions, were carried out as before [3]. Western blotting was performed according to [17,18].…”