Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Voltage-gated sodium channels are molecular targets of β-scorpion toxins, which enhance excitability by shifting the voltage dependence of activation to more negative potentials. These effects result from a voltage sensor trapping mechanism, in which toxins trap the voltage sensor in its activated conformation. Determinants of β-scorpion toxin (CssIV) binding and action on sodium channel (Nav1.2) are located in the S1-S2 and S3-S4 extracellular linkers in the voltage-sensing module in domain II. To completely map these regions, we made substitutions for previously unstudied amino acid residues and examined modulation by CssIVE15A, a highly active toxin derivative. Of 11 positions studied in IIS1-S2, only one significantly altered the toxin effect from wild-type by reducing binding to the resting state and almost abolishing trapping activity. In IIS3-S4, five positions surrounding a previously identified key binding determinant, G845, define a hotspot of high impact residues. Three of these substitutions reduced toxin binding and voltage-sensor trapping. The other two, V843A and E844N, increased voltage-sensor trapping approximately 4-fold and decreased apparent EC50. The rate of voltage sensor trapping upon depolarization was unchanged for V843A and increased approximately 2.5-fold for E844N. The rate at which the toxin releases the voltage sensor upon repolarization was increased 2.2-fold for the V843A but was unchanged for E844N. Thus CssIVE15A interacts with a short segment of IIS1-S2 and a broader region of DIIS3-S4. The bidirectional effects of mutations on toxin efficacy suggest that native residues make both positive and negative interactions with the toxin. Substitutions that increase toxin effects do so by increasing affinity of resting channels for the toxin and further increasing the relative affinity of the activated voltage-sensor for the toxin. These results provide further support for the voltage sensor-trapping model.