Binding of [³H] γ‐Aminobutyric Acid and [³H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract

Abstract: The binding of [ 3 H] γ ‐aminobutyric acid ([ 3 H]GABA) and [ 3 H]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 μM γ ‐aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K D and B max values for [ 3 H]GABA at 4°C were K D1 , 20 n M ; K D2 ,165 n M; B max1 , 0.48 pmol; B max2 , 6.0 pmol. mg −1 ; for [ 3 H]muscimol at 4°C they were: K D1 , 1.75 n M; K D2 , 17.5 n M; B maxl , 0.84 pmol. mg −1 ; B max2 , 4.8 pmol.mg −1 ; and for [ 3 H]muscimol at 37°C they were: K D1 , 7.0 nM; K m , 60 nM; B max] , 0.5 pmol‐mg −1 ; B max2 , 7.2 pmol‐mg 1 . Under the experimental conditions used, the similar B milx values for [ 3 H]GABA and [ 3 H]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 [ 3 H]muscimol at 4 C C 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 K D values obtained suggest a pA 2 value of 5.3 against [ 3 H]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 [ 3 H]GABA and [ 3 H]muscimol suggest that [ 3 H]GABA might be a more satisfactory ligand than [ 3 H]muscimol in GABA radioreceptor assays.