Photoaffinity labeling of the GABAA receptor with [3H]muscimol.

Article date: 1985/3/1

PubMed ID: 2857769

Journal name: Journal of neurochemistry (ISSN: 0022-3042)


Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).

This document is available from: http://directlinks.cc/files/muscimol/2857769.pdf

Author List: Cavalla D, Neff N H

Publication Types: Journal Article

Substances mentioned in the article: Affinity Labels; Glutamates; Oxazoles; Receptors, GABA-A; Muscimol; Glutamic Acid; Baclofen; Diazepam;

Mesh terms: Affinity Labels/metabolism; Animals; Baclofen/pharmacology; Diazepam/pharmacology; Glutamates/pharmacology; Glutamic Acid; Kinetics; Male; Molecular Weight; Muscimol/metabolism; Oxazoles/metabolism; Photochemistry; Rats; Rats, Inbred Strains; Receptors, GABA-A/metabolism; Spectrophotometry, Ultraviolet;

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