Identification of a unique domain in bovine brain GABAA receptors that is photoaffinity labelled by [3H]Ro15-4513

We have used photoaffinity labelling and protein cleavage techniques to identify the site of photoincorporation of [3H]Ro15-4513 into the alpha subunit of the bovine gamma-aminobutyric acid type A (GABAA) receptor. Bovine brain membranes were photoaffinity labelled with [3H]Ro15-4513 and after solubilization and denaturation, proteins were specifically cleaved at either cysteine or tryptophan residues. Peptides were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Cleavage at cysteine residues generated a labelled peptide of Mr 6.5K, while cleavage at tryptophan residues generated a labelled peptide with an Mr of 5K. Cleavage products of this size indicate that the site of [3H]Ro15-4513 incorporation occurs between the end of the first transmembrane domain and the first four amino acids of the third transmembrane domain (residues 247-289). This region of the GABAA receptor has not previously been implicated in the formation of the benzodiazepine binding site and may be part of a unique recognition domain for inverse agonists.