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

Abstract

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 [³H]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 [³H]SR 95531 for its binding site; Na⁺ had no effect. Subcellular fractionation showed that 74% of the P₂ binding was in synaptosomes; 31% of the total homogenate binding was in P₂ and 50% in P₃. The binding of [³H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (K_D of 6.34 nM and B_max of 0.19 pmol/mg of protein; K_D of 32 nM and B_max of 0.81 pmol/mg of protein). The binding of [³H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABA_A ligands were effective displacers of [³H]SR 95531. GABA_A antagonists were relatively more potent in displacing [³H]SR 95531 than [³H]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 [³H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABA_A receptor site in its antagonist conformation.