The bioactive surface of scorpion -toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved ␣ core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian -toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated -toxins (⌬⌬Bj-xtrIT and ⌬⌬Css4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, ⌬⌬Bj-xtrIT and ⌬⌬Css4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion ␣-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion ␣-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of Lqh␣IT at insect sodium channels. Co-application of ⌬⌬Bj-xtrIT or ⌬⌬Css4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at ⌬⌬Bj-xtrIT and ⌬⌬Css4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both -toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.Voltage-gated sodium channels (Na v s) 3 are critical for generation and propagation of action potentials in excitable cells and are targeted by a large variety of ligands that bind at distinct receptor sites on the pore-forming ␣-subunit (1). Most lipidsoluble Na v activators, including pyrethroid insecticides, toxic alkaloids (e.g. veratridine, from the plant family of Liliaceae, and batrachotoxin, from the skin of the Colombian frog Phyllobates aurotaenia), and marine cyclic polyether toxins (e.g. brevetoxins produced by "red tide" dinoflagellates), are effective in invertebrates as well as vertebrates. In addition, a wide array of proteinaceous Na v modifiers exists in the venom of scorpions, spiders, cone snails, and sea anemones, which are utilized for prey and defense (1, 2). Na v gating modifiers from scorpion venom are divided into two classes, ␣ and , according to their mode of action and binding properties (3-5). ␣-Toxins prolong the action potential by inhibiting the fast inactivation of the Na v upon binding to receptor site-3 (4, 6), which involves extracellular loops in domains 1 and 4 (6). These toxins are divided into three groups (7): (i) anti-mammalian ␣-toxins (e.g. Lqh2 from Leiurus quinquestriatus hebraeus), which are highly toxic in mammalian brain and bind with high affinity to rat brain Na v s; (ii) antiinsect ␣-toxins (e.g. Lqh␣IT), which are highly toxic to insects and show weak activity in mammalian brain; and (iii) ␣-like toxins (e.g. Lqh3), ...