The interaction of insect-selective scorpion depressant β-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, 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 BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 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, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. 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.

中文翻译：

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用昆虫钠通道绘制蝎子 β-毒素的相互作用表面

昆虫选择性蝎子抑制剂 β-毒素（LqhIT2 和来自 Leiurus quinquestriatus hebraeus 的 Lqh-dprIT3）与德国小蠊钠通道 BgNav1-1a 的相互作用使用定点诱变、电生理分析和结构模型进行了研究。关注电压感应域 II (VSD-II) 处蝎子 β-毒素的药理学定义的结合位点 4，我们发现 VSD-II 中 D802 的电荷中和大大增强了对 Lqh-dprIT3 的通道敏感性。这与带有 G802 的剪接变体 BgNav2-1 对 Lqh-dprIT3 的高敏感性以及 BgNav2-1 突变体 G802D 对毒素的低敏感性一致。进一步的突变和电生理分析表明，WT = D802E < D802G < D802A < Lqh-dprIT3 的 D802K 通道突变体与无毒素通道中激活的去极化转变相关。然而，涉及 IIS4 碱性残基（K4E = WT << R1E < R2E < R3E）或双突变体（D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT）的单突变体的敏感性不相关随着激活转变。使用美洲大蠊通道 NavPaS 的冷冻电镜结构作为模板和 LqhIT2 的晶体结构，我们构建了与 BgNav1-1a 复合的 LqhIT2 和 Lqh-dprIT3-c 的结构模型。这些模型以及突变分析表明，抑制毒素在 VSD-II 处接近 R1 和 D802 之间的盐桥，与接头 IIS1-S2、IIS3-S4、IIIP5-P1 和 IIIP2-S6 形成接触。