title: Transmembrane topology of the gamma-aminobutyric acidA/benzodiazepine receptor: subcellular distribution and allosteric coupling determined in situ.
abstract: The subcellular distribution, allosteric coupling, and topology of the gamma-aminobutyric acidA (GABA)/benzodiazepine receptor were investigated in situ. We have shown previously that a transmembrane fragment (Mr = 24,000) of the benzodiazepine receptor remains in the plasma membrane after trypsin treatment of embryonic brain neurons maintained in primary monolayer cell culture. Here we report a study of the effects of exhaustive trypsinization on the binding of ligands to the GABA recognition site. Approximately 60% of the binding sites for [3H]muscimol in intact cells are inactivated by extracellular trypsin and, therefore, are associated with cell surface GABA receptors, whereas 40% of the sites are trypsin resistant. GABA potentiates [3H]flunitrazepam binding to intact cells and trypsin-treated intact cells. GABA-induced enhancement of [3H]flunitrazepam binding to intact cells is eliminated when cell-surface benzodiazepine receptors are blocked with Ro7-0213 (a charged benzodiazepine), but some enhancement remains after extracellular trypsinization. This provides indirect evidence for the existence of a population of trypsin-resistant GABA recognition sites associated with cell-surface receptors and indicates that trypsin-resistant cell-surface GABA recognition sites are allosterically linked to trypsin-resistant cell-surface benzodiazepine recognition sites, which we have shown previously to be associated with the trypsin-generated Mr 24,000 transmembrane fragment. The results are discussed in terms of a model of the transmembrane topology of the GABA/benzodiazepine receptor.
authors.affiliation: Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn 11203.
authors.forename: C
authors.initials: C
authors.lastname: Czajkowski
authors.forename: T T
authors.initials: TT
authors.lastname: Gibbs
authors.forename: D H
authors.initials: DH
authors.lastname: Farb
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology; D002462:Cell Membrane / Q000032:analysis; D002478:Cells, Cultured; D002642:Chick Embryo; D005445:Flunitrazepam / Q000378:metabolism; D009118:Muscimol / Q000378:metabolism; D009474:Neurons / Q000032:analysis; D011963:Receptors, GABA-A / Q000032:analysis*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D011963:Receptors, GABA-A; C050150:Ro 7-0213; D001569:Benzodiazepines; D009118:Muscimol; D005445:Flunitrazepam; D014357:Trypsin