Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific γ-aminobutyric acidA-receptor antibodies

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