IDENTIFICATION OF DOMAINS IN HUMAN RECOMBINANT GABA(A) RECEPTORS THATARE PHOTOAFFINITY-LABELED BY [H-3] FLUNITRAZEPAM AND [H-3] RO15-4513

We have used [H-3]flunitrazepam and [H-3]Ro15-4513 as photoaffinity la belling agents in combination with a chemical cleavage technique to lo calize the benzodiazepine recognition sites of specific human recombin ant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABA(A) receptor subtypes. The chemical agent utilized was hyd roxylamine, whose substrate is a relatively rare asparagine-glycine am ide bond that occurs only in the alpha subunits of the receptors exami ned in this study. Cleavage products were resolved using sodium dodecy l sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing rece ptors, incorporation of [H-3]flunitrazepam occurs within residues 1-10 3 of the alpha 1 subunit, while incorporation of [H-3]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 10 4 and the C-terminus. Photolabelling of membranes prepared from the al pha 6 beta 3 gamma 2-expressing cell line with [H-3]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r)56 000 and M(r) 48 000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [H-3]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [H-3]flunitrazepam and the inverse a gonist [H-3]Ro15-4513 occur within distinct domains of the human GABAA receptor. Copyright (C) 1996 Elsevier Science Ltd