GABA-stimulated 36Cl − influx into reconstituted vesicles with purified GABA A/benzodiazepine receptor complex

Abstract

Solubilized and Purified γ-aminobutyric acid (GABA) A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl − influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl − influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABA A/benzodiazepine receptor complex and Cl − channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl − channel also exhibited GABA-stimulated 36Cl − influx. This GABA-stimulated influx of 36Cl − was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABA A receptor antagonist. These results strongly suggest that GABA A receptor is directly coupled with Cl − channel, whereas benzodiazepine receptor may be functionally coupled with GABA A receptor and modulates the GABA-stimulated Cl − influx through GABA A receptor. The present results also indicate that the purified GABA A receptor complex is coupled with Cl − channel and possesses functional characteristics as GABA A receptor.