Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific γ-aminobutyric acid(A)-receptor antibodies

Abstract

The γ-aminobutyric acid(A) (GABA(A)) receptor purified from adult bovine cerebral cortex was photpaffinity-labelled with the agonist benzodiazepine [3H]flunitrazepam and the radioactivity shown to be coincident with a band with M(r) 53,000 that was recognized by three anti-(GABA(A) receptor α1 subunit sequence)-specific antibodies. Complete and limited CNBr cleavage of the purified photoaffinity-labelled receptor was carried out. The products of this reaction were analysed for radioactivity, for immunoreactivity with anti-[α1-(1-15)-peptide], anti-[α1-(324-341)-peptide] and anti-[α1-(413-429)-peptide] polyclonal antibodies and for carbohydrate by biotinylated concanavalin A lectin overlay. Complete CNBr cleavage gave a radioactive peptide with M(r) 10,000-12,000 that was not recognized by the above-mentioned specific antisera. By using the deduced amino acid sequence of the α1 subunit [Schofield, Darlison, Fujita, Burt, Stephenson, Rodriguez, Rhee, Ramachandra, Reale, Glencorse, Seeburg and Barnard (1987) Nature (London) 328, 221-227], it is proposed that the site of the benzodiazepine-agonist photoaffinity-labelling reaction does not lie within the amino acid sequences α1 1-58 and α1 149-429.