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A novel GABAA antagonist [3H]SR 95531: Microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands

Recent reports have demonstrated that the synthetic gamma‐aminobutyric acid (GABA)‐derivative, SR 95531 [2‐(3′‐carbethoxy‐2′‐propyl)‐3‐amino‐6‐paramethoxy‐phenyl‐pyridazini‐um bromide], possesses selective GABAA sites, this agent has been used to identify GABAA receptors. In the present investigation, we studied the binding of [3H]SR 95531 to tissue sections of rat brain using microscopic analysis of receptor localization. The appropriate binding conditions for defining GABAA receptors with this radioligand were obtained by determining the dissociation and association kinetics, and performing saturation and displacement studies. Using membrane preparations from whole rat brain (or brain regions representing cortex, striatum, hippocampus, midbrainthalamus, medulla‐pons and cerebellum), saturation and displacement studies were analyzed, and allosteric modulation of [3H]SR 95531 binding was examined by including several benzodiazepine and barbiturate receptor ligands in the incubation media. To assess the stereoselective properties of [3H]SR 95531 binding in rat membranes, numerous barbiturates were added during the incubation. The binding of [3H]SR 95531 was demonstrated to be saturable, specific and to bind with relatively high affinity to low‐affinity GABAA sites. Scatchard analysis performed on saturation data of binding to tissue sections showed a dissociation constant (KD) of 42.4 nM and a maximum number of binding sites (Bmax) of 105.8 fmol/mg tissue. Microscopic analysis showed that intermediate to high densities of [3H]SR 95531 binding occurred in brain regions containing intermediate to high densities of low‐affinity GABAA sites. Results from competition studies demonstrated that [3H]SR 95531 is displaceable by GABAA agents and displaced preferentially by GABAA antagonists. Scatchard analysis of saturation experiments from membrane preparations indicated that the KD and Bmax from the centrifugation assay was 53.0 nM and 4.26 pmol/mg protein, respectively. Using the filtration assay, binding to membranes yielded a KD value of 45.6 nM and a Bmax of 0.77 pmol/mg protein. The allosteric modulation data demonstrated that numerous benzodiazepine and barbiturate agents inhibited [3H]SR 95531 binding and this varied according to brain region. Several barbiturates included in the incubation media exhibited a stereoselective inhibition of [3H]SR 95531 binding to whole rat brain membranes. In summary, [3H]SR 95531 identifies the antagonist conformation of GABAA receptors in the rat brain and binds preferentially to the low‐affinity site. The data collected in the present study show that [3H]SR 95531 is an excellent probe to recognize the GABAA receptor subtype and allows for the identification of the interactions of subcomponents of the macromolecular GABA receptor complex.