Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind ␣-and ␣-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with ␣-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled ␣-scorpion toxins AaH II and Lqh␣IT, the most active ␣-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The ␣-mammal scorpion toxins and the anti-insect Lqh␣IT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with ␣-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and Lqh␣IT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster.

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Scorpion ␣-toxins are similar in their mode of action and three-dimensional structure but differ considerably in affinity for various voltage-gated sodium channels (NaChs). To clarify the molecular basis of the high potency of the ␣-toxin Lqh␣IT (from Leiurus quinquestriatus hebraeus) for insect NaChs, we identified by mutagenesis the key residues important for activity. We have found that the functional surface is composed of two distinct domains: a conserved "Core-domain" formed by residues of the loops connecting the secondary structure elements of the molecule core and a variable "NC-domain" formed by a five-residue turn (residues 8 -12) and a C-terminal segment (residues 56 -64). We further analyzed the role of these domains in toxin activity on insects by their stepwise construction onto the scaffold of the anti-mammalian ␣-toxin, Aah2 (from Androctonus australis hector). The chimera harboring both domains, Aah2Lqh␣IT(face) , was as active to insects as Lqh␣IT. Structure determination of Aah2Lqh␣IT(face) by x-ray crystallography revealed that the NC-domain deviates from that of Aah2 and forms an extended protrusion off the molecule core as appears in Lqh␣IT. Notably, such a protrusion is observed in all ␣-toxins active on insects. Altogether, the division of the functional surface into two domains and the unique configuration of the NC-domain illuminate the molecular basis of ␣-toxin specificity for insects and suggest a putative binding mechanism to insect NaChs.Voltage-gated sodium channels (NaChs) 1 mediate the transient increase in sodium ion permeability that triggers action potentials in excitable cells (1). These channels are composed of a pore-forming ␣-subunit (260 kDa) associated with one or two auxiliary ␤-subunits. The ␣-subunit consists of four repeat domains (D1-D4), each containing six transmembrane segments (S1-S6) and a membrane-associated re-entrant segment (SS1-SS2), connected by internal and external loops. A key feature in the function of NaChs is their gating behavior, namely the ability to rapidly activate and inactivate upon cell membrane depolarization, leading to transient increase in Na ϩ conductance (1). Due to their key role in excitability, these channels are targeted by a variety of toxins.Long-chain scorpion toxins are 61-to 76-residue-long polypeptides that share a similar core composed of an ␣-helix packed against a three-stranded ␤-sheet and stabilized by four disulfide bonds. These toxins bind to various receptor sites on the extracellular face of NaChs and alter their gating. Traditionally, they are divided into two major classes, ␣-and ␤-toxins, according to their mode of action and binding properties to distinct receptor sites on NaChs (2, 3).Scorpion ␣-toxins prolong the action potential by slowing channel inactivation, possibly through interference with the outward movement of the D4S4 segment necessary for the fast inactivation process (4). The scorpion ␣-toxin binding site, termed neurotoxin receptor site-3, has been shown to involve the extracellular regions of D...

Scorpion ␤-toxins that affect the activation of mammalian voltage-gated sodium channels (Na v s) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion ␤-toxin, Css4, whose effects on rat brain Na v s have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 Å 2 composed of the following: 1) a cluster of residues associated with the ␣-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion ␤-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the ␤2 and ␤3 strands, which is likely to confer the specificity for mammalian Na v s; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion ␤-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.