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Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

ABSTRACT: Scorpion ?-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.

SUBMITTER: Karbat I

PROVIDER: S-EPMC2945547 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

Karbat Izhar I Ilan Nitza N Zhang Joel Z JZ Cohen Lior L Kahn Roy R Benveniste Morris M Scheuer Todd T Catterall William A WA Gordon Dalia D Gurevitz Michael M

The Journal of biological chemistry 20100803 40

Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased an ...[more]

PMID: 20682774

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Project description:?-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 ?-toxins comprising 13 members (named MeuNaTx?-1 to MeuNaTx?-13) from the scorpion Mesobuthus eupeus. Of them, five native toxins (MeuNaTx?-1 to -5) were purified to homogeneity from the venom and the solution structure of MeuNaTx?-5 was solved by nuclear magnetic resonance. A systematic functional evaluation of MeuNaTx?-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?-4 shows no activity on these isoforms and thus represents the first Mesobuthus-derived insect-selective ?-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?-2 only affects Na(v)1.4; MeuNaTx?-1 and MeuNaTx?-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?-4 and MeuNaTx?-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 ?-toxins. The PSSs include site 50 and other seven sites located in functional surfaces of ?-toxins. This work represents the first thorough investigation of evolutionary diversification of ?-toxins derived from a specific scorpion lineage from the perspectives of sequence, structure, function, and evolution.

Dataset Information

Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

Publications

Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

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