Common features in the functional surface of scorpion β-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion β-toxins that affect the activation of mammalian voltage-gated sodium channels (Na_vs) 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 β-toxin, Css4, whose effects on rat brain Na_vs 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 Ų composed of the following: 1) a cluster of residues associated with the α-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion β-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the β2 and β3 strands, which is likely to confer the specificity for mammalian Na_vs; 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 β-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.