Scorpion beta and alpha-toxins modify the activation and inactivation of voltage-gated sodium channels. Although the two types of toxin bind at two distinct receptor sites on the same sodium channel, they exhibit synergic effects when coinjected into insects. To clarify the basis of this synergism we examined the mutual effects of alpha and beta toxin representatives in radio-ligand binding assays. We found positive allosteric interactions between receptor site-4 of the excitatory Bj-xtrIT and the depressant LqhIT2 beta toxins and receptor site-3 of the alpha toxin LqhIT, on locust neuronal membranes. Unexpectedly, a nontoxic mutant Bj-xtrIT-E15R, which binds with high affinity to receptor site-4, was able to enhance LqhIT binding and toxicity similarly to the unmodified Bj-xtrIT. This result indicates that mere binding of a nontoxic ligand to receptor site-4 ("silent binding") induces a conformational change that does not alter channel gating, but influences toxin binding at receptor site-3 leading to enhanced toxicity. This finding suggests a new functional role for weakly toxic polypeptides in that they enhance the effect of other active neurotoxins in the arthropod venom. Such silent binding may have also valuable implications in attempts to improve drug efficacy by combining potent drugs with nonactive allosteric enhancers.--Cohen, L., Lipstein, N., Gordon, D. Allosteric interactions between scorpion toxin receptor sites on voltage-gated Na channels imply a novel role for weakly active components in arthropod venom.