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

Journal Article · Tue Mar 21 00:00:00 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), Vol. 28:6; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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