Abstract

α-Dihydropicrotoxinin, which has a pharmacological activity similar to that of picrotoxin in producing convulsions and inhibiting the synaptic responses of γ-aminobutyric acid in arthropod muscle and the vertebrate central nervous system, was investigated as a possible probe for characterizing γ-aminobutyric acid receptor-ionophore function in mammalian brain. [³H]α-Dihydropicrotoxinin was synthesized and found to bind rapidly, reversibly, and in a saturable fashion to particulate fractions of rat brain homogenates, with an apparent K_D of 1-2 µM. The binding sites showed a similar subcellular as well as brain regional distribution to presumed γ-aminobutyric acid receptor binding sites, consistent with a postsynaptic membrane location. The density of dihydropicrotoxinin binding sites was about 5 pmoles/mg protein, or 130 ± 20 pmoles/g of wet brain, which is within a factor of 2 of the number of γ-aminobutyric acid receptor sites. Six picrotoxin analogues showed an excellent correlation between convulsant activity and potency in inhibiting binding, and some other convulsant and anticonvulsant drugs inhibited the binding, suggesting that the binding sites may be related to the pharmacological effects. γ-Aminobutyric acid (up to 1 mM) and muscimol (0.1 mM) did not affect the binding. Since picrotoxinin has likewise been found not to inhibit γ-aminobutyric acid binding to receptor sites in brain, the results support the interpretation that picrotoxinin inhibits γ-aminobutyric acid synapses by binding at a site distinct from the γ-aminobutyric acid recognition site. The picrotoxinin binding sites are likely to be related to macromolecules that regulate chloride permeability and translate the γ-aminobutyric acid recognition site (receptor) interaction into the physiological response.