Abstract

[³H]α-Dihydropicrotoxinin, a biologically active analogue of the γ-aminobutyric acid synaptic antagonist picrotoxin, was found to bind to a small quantity of saturable, high-affinity (K_D, 1 µM) sites in crayfish muscle homogenates. Dihydropicrotoxinin binding was rapid, reversible, and proportional to protein concentration. This binding, like that of γ-aminobutyric acid, was enriched in sarcolemma fractions, being present at 6-8 pmoles/mg of protein or per gram of wet muscle. The quantity of binding sites varied with muscle types in a manner consistent with varying degrees of inhibitory innervation. The binding was inhibited by picrotoxinin analogues in the same order of potency shown by these compounds in convulsant activity or in inhibition of γ-aminobutyric acid synaptic responses: picrotoxinin and tutin were slightly more active than dihydropicrotoxinin in all systems, whereas picrotin was less active and picrotoxinin acetate and alkali-hydrolyzed picrotoxinin were inactive. γ-Aminobutyric acid up to 0.2 mM did not inhibit dihydropicrotoxinin binding. α-Dihydropicrotoxinin binding sites appear to be related to the physiological action of the drug, and this radioactive toxin may provide a useful probe for postsynaptic membrane macromolecules that regulate the inhibitory chloride ionophore at sites distinct from the γ-aminobutyric acid receptor.