Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol

Abstract

Muscimol is one of the most potent agonist ligands at the 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 [maximum binding capacity] of reversible [3H]muscimol binding. 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 3 compounds was characteristic of labeling to the GABAA receptor. Baclofen, 1-glutamate and diazepam exerted no effect at high concentrations. SDS-PAGE [sodium dodecyl sulfate-polyacrylamide gel electrophoresis] of the photolabeled membranes indicated specific incorporation of radioactivity into 2 MW species. One failed to enter the separating gel, implying a MW > 250,000 daltons (250 kD). The MW of the other was identified by fluorography to be .apprx. (52 kD).