Fluorescence measurements of anion transport by the GABAA receptor in reconstituted membrane preparations

Abstract

A fluorescence assay for measuring the functional properties of the GABAA receptor in reconstituted membrane vesicles is described. This assay is based on a method previously described to measure monovalent cation transport mediated by the nicotinic acetylcholine receptor in membranes from Torpedo electric organ [Moore, H.-P.H., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4509-4513]. The GABAA receptor has been solubilized from bovine brain membranes and reconstituted into phospholipid vesicles. Influx of chloride or iodide into the vesicles has been measured in stopped-flow experiments by monitoring the fluorescence quench of an anion-sensitive fluorophore trapped within the vesicles. Muscimol, a GABAA receptor agonist, stimulated a rapid uptake of either chloride or iodide. Stimulation of chloride influx was dependent on the concentration of muscimol, and the midpoint of the dose-response curve occurred at approximately 0.3 microM. Agonist-stimulated uptake was enhanced by diazepam and blocked by desensitization and by the antagonists bicuculline and picrotoxin. These receptor-mediated effects are shown to be qualitatively similar to measurements of 36Cl- and 125I- efflux using synaptoneurosomes prepared from rat cerebral cortex. The advantages of the fluorescence method in terms of its improved time resolution, sensitivity, and suitability for quantitating GABAA receptor function are discussed.