Evolutionary Diversification of Mesobuthus α-Scorpion Toxins Affecting Sodium Channels

alpha-Scorpion toxins constitute a family of peptide modulators that induce a prolongation of the action potential of excitable cells by inhibiting voltage-gated sodium channel inactivation. Although they all adopt a conserved structural scaffold, the potency and phylogentic preference of these toxins largely vary, which render them an intriguing model for studying evolutionary diversification among family members. Here, we report molecular characterization of a new multigene family of alpha-toxins comprising 13 members (named MeuNaTx alpha-1 to MeuNaTx alpha-13) from the scorpion Mesobuthus eupeus. Of them, five native toxins (MeuNaTx alpha-1 to -5) were purified to homogeneity from the venom and the solution structure of MeuNaTx alpha-5 was solved by nuclear magnetic resonance. A systematic functional evaluation of MeuNaTx alpha-1, -2, -4, and -5 was conducted by two-electrode voltage-clamp recordings on seven cloned mammalian voltage-gated sodium channels (Na(v)1.2 to Na(v)1.8) and the insect counterpart DmNa(v)1 expressed in Xenopus oocytes. Results show that all these four peptides slow inactivation of DmNa(v)1 and are inactive on Na(v)1.8 at micromolar concentrations. However, they exhibit differential specificity for the other six channel isoforms (Na(v)1.2 to Na(v)1.7), in which MeuNaTx alpha-4 shows no activity on these isoforms and thus represents the first Mesobuthus-derived insect-selective alpha-toxin identified so far with a half maximal effective concentration of 130 +/- 2 nM on DmNa(v)1 and a half maximal lethal dose of about 200 pmol g(-1) on the insect Musca domestica; MeuNaTx alpha-2 only affects Na(v)1.4; MeuNaTx alpha-1 and MeuNaTx alpha-5 have a wider range of channel spectrum, the former active on Na(v)1.2, Na(v)1.3, Na(v)1.6, and Na(v)1.7, whereas the latter acting on Na(v)1.3-Na(v)1.7. Remarkably, MeuNaTx alpha-4 and MeuNaTx alpha-5 are two nearly identical peptides differing by only one point mutation at site 50 (A50V) but exhibit rather different channel subtype selectivity, highlighting a switch role of this site in altering the target specificity. By the maximum likelihood models of codon substitution, we detected nine positively selected sites (PSSs) that could be involved in functional diversification of Mesobuthus alpha-toxins. The PSSs include site 50 and other seven sites located in functional surfaces of alpha-toxins. This work represents the first thorough investigation of evolutionary diversification of alpha-toxins derived from a specific scorpion lineage from the perspectives of sequence, structure, function, and evolution. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.012054, 1-18, 2012.