TRANSMEMBRANE TOPOLOGY OF THE GAMMA-AMINOBUTYRIC ACIDA BENZODIAZEPINE RECEPTOR - SUBCELLULAR-DISTRIBUTION AND ALLOSTERIC COUPLING DETERMINED INSITU

Google Scholar
  • 1 January 1989
    • journal article
    • research article
    • Vol. 35  (1) , 75-84
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 of 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.