Binding of [3H]gamma-aminobutyric acid and [3H]muscimol in purified rat brain synaptic plasma membranes and the effects of bicuculline.

Abstract

Abstract: The binding of [3H] γ‐aminobutyric acid ([3H]GABA) and [3H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ‐aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent KD and Bmax values for [3H]GABA at 4°C were KD1, 20 nM; KD2,165 nM; Bmax1, 0.48 pmol;Bmax2, 6.0 pmol. mg−1; for [3H]muscimol at 4°C they were: KD1, 1.75 nM; KD2, 17.5 nM; Bmaxl, 0.84 pmol. mg−1; Bmax2, 4.8 pmol.mg−1; and for [3H]muscimol at 37°C they were: KD1, 7.0 nM; Km, 60 nM; Bmax], 0.5 pmol‐mg−1; Bmax2, 7.2 pmol‐mg1. Under the experimental conditions used, the similar Bmilx values for [3H]GABA and [3H]muscimol binding to the SPM preparations suggests that the high‐ and low‐affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [3H]muscimol at 4CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high‐affinity site but noncompetitive at the low‐affinity site. At 37°C, antagonism was again competitive at the high‐affinity site but was of a mixed competitive/noncompetitive nature at the low‐affinity site. Assuming that binding to the high‐affinity site is associated with the pharmacological actions of bicuculline, the apparent KD values obtained suggest a pA2 value of 5.3 against [3H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [3H]GABA and [3H]muscimol suggest that [3H]GABA might be a more satisfactory ligand than [3H]muscimol in GABA radioreceptor assays.