title: Photoaffinity labeling of the GABAA receptor with [3H]muscimol.
abstract: 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).
authors.forename: D
authors.initials: D
authors.lastname: Cavalla
authors.forename: N H
authors.initials: NH
authors.lastname: Neff
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism*; D010080:Oxazoles / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000345:Affinity Labels; D005971:Glutamates; D010080:Oxazoles; D011963:Receptors, GABA-A; D009118:Muscimol; D018698:Glutamic Acid; D001418:Baclofen; D003975:Diazepam