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

Journal Article · Tue Mar 21 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00432a031· OSTI ID:5478564

Dunn, S M.J.; Shelman, R A; Agey, M W ^[1]

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1. Univ. of Iowa, Iowa City (USA)

A fluorescence assay for measuring the functional properties of the GABA{sub A} 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. The GABA{sub A} 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 GABA{sub A} 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 {mu}M. 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 {sup 36}Cl{sup {minus}} and {sup 125}I{sup {minus}} 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 GABA{sub A} receptor function are discussed.

• OSTI ID: 5478564
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:6; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AMINOBUTYRIC ACID
ANIMAL CELLS
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOELECTRICITY
BODY
BRAIN
CARBOXYLIC ACIDS
CATTLE
CENTRAL NERVOUS SYSTEM
CHLORINE 36
CHLORINE ISOTOPES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
DRUGS
ELECTRICITY
ELECTRON CAPTURE RADIOISOTOPES
FLUORESCENCE
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LIGANDS
LIGHT NUCLEI
LUMINESCENCE
MAMMALS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
NERVE CELLS
NERVOUS SYSTEM
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
RUMINANTS
SOMATIC CELLS
UPTAKE
VERTEBRATES
YEARS LIVING RADIOISOTOPES