Mapping the interaction surface of scorpion β-toxins with an insect sodium channel

The interaction of insect-selective scorpion depressant β-toxins (LqhIT2 and Lqh-dprIT₃ from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNa_v1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion β-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT₃. This was consistent with the high sensitivity of the splice variant BgNa_v2-1, bearing G802, to Lqh-dprIT₃, and low sensitivity of BgNa_v2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT₃ correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, Na_vPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT₃-c in complex with BgNa_v1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1–S2, IIS3–S4, IIIP5–P1 and IIIP2–S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT₃.