Transmembrane topology of the gamma-aminobutyric acidA/benzodiazepine receptor: subcellular distribution and allosteric coupling determined in situ.

Article date: 1989/1/1

PubMed ID: 2536471

Journal name: Molecular pharmacology (ISSN: 0026-895X)


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.

Author List: Czajkowski C, Gibbs T T, Farb D H

Publication Types: Journal Article; Research Support, U.S. Gov't, P.H.S.

Substances mentioned in the article: Receptors, GABA-A; Ro 7-0213; Benzodiazepines; Muscimol; Flunitrazepam; Trypsin;

Mesh terms: Allosteric Regulation; Animals; Benzodiazepines/pharmacology; Cell Membrane/analysis; Cells, Cultured; Chick Embryo; Flunitrazepam/metabolism; Muscimol/metabolism; Neurons/analysis; Receptors, GABA-A/analysis; Trypsin/pharmacology;

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