title: Identification of domains in human recombinant GABAA receptors that are photoaffinity labelled by [3H]flunitrazepam and [3H]Ro15-4513.
abstract: We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]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 [3H]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 [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Davies
authors.forename: I L
authors.initials: IL
authors.lastname: Martin
authors.forename: A N
authors.initials: AN
authors.lastname: Bateson
authors.forename: K L
authors.initials: KL
authors.lastname: Hadingham
authors.forename: P J
authors.initials: PJ
authors.lastname: Whiting
authors.forename: S M
authors.initials: SM
authors.lastname: Dunn
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000493:pharmacokinetics*; D001569:Benzodiazepines / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005445:Flunitrazepam / Q000493:pharmacokinetics*; D018755:GABA Agonists / Q000494:pharmacology; D018757:GABA Modulators / Q000493:pharmacokinetics*; D006801:Humans; D006898:Hydroxylamines; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D001386:Azides; D018755:GABA Agonists; D018757:GABA Modulators; D006898:Hydroxylamines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D005445:Flunitrazepam; C042957:Ro 15-4513