Characterization of the Binding of [ 3 H]SR 95531, a GABA A Antagonist, to Rat Brain Membranes

A synthetic derivative of γ‐aminobutyric acid (GABA), SR 95531 [2‐(3′‐carboxy‐2′‐propyl)‐3‐amino‐6‐ p ‐methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microion‐tophoretic studies, to be a potent, selective, competitive, and reversible GABA A antagonist. In the present study, the binding of [ 3 H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4°C after 30 min of incubation. Pretreatment of the membranes with Triton X‐100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [ 3 H]SR 95531 for its binding site; Na + had no effect. Subcellular fractionation showed that 74% of the P 2 binding was in synaptosomes; 31% of the total homogenate binding was in P 2 and 50% in P 3 . The binding of [ 3 H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites ( K D of 6.34 n M and B max of 0.19 pmol/mg of protein; K D of 32 n M and B max of 0.81 pmol/mg of protein). The binding of [ 3 H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABA A ligands were effective displacers of [ 3 H]SR 95531. GABA A antagonists were relatively more potent in displacing [ 3 H]SR 95531 than [ 3 H]GABA; the inverse was true for GABA A agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex > thalamus = olfactory bulb = hypothala‐mus = amygdala = striatum > pons‐medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of B max values at both the high‐ and the low‐affinity binding sites. In conclusion, the present results demonstrate specific, high‐affinity, saturable, and reversible binding of [ 3 H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABA A receptor site in its antagonist conformation.