Department of Plant Molecular Biology & Ecology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
Toxicon. 2012 Sep 15;60(4):502-11. doi: 10.1016/j.toxicon.2012.03.022. Epub 2012 Apr 4.
Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain II, while Lqh2 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain IV. Double-mutant cycle analysis and dissociation assays employing a battery of Lqh2 mutants against rNa(v)1.2a mutants identified the docking orientation of alpha toxins at the channel external surface of the Gating-module in domain IV. The other point of interaction between the toxin and the channel has not yet been defined and may involve channel residues of either the Pore-module or the Gating-module.
蝎类 alpha 和 beta 毒素与电压门控钠离子通道 (Na(v)s) 在两个药理学上不同的位点相互作用。alpha 毒素结合在受体 site-3 上并抑制通道失活，而 beta 毒素结合在受体 site-4 上并将电压依赖性激活移向更超极化的电位。这两种毒素类别根据其结合偏好和竞争 Na(v)亚型受体位点的能力被细分为不同的药理学组。为了阐明两个受体位点的毒素-通道相互作用表面，并阐明不同毒素偏好的分子基础，建立了一种用于在重组形式中表达它们的高效细菌系统。伴随毒性、结合和电生理测定的突变，以及使用 NMR 和 X 射线晶体学确定三维结构，揭示了所有药理学组毒素代表物中存在一个二分的生物活性表面。在哺乳动物脑通道 rNa(v)1.2a 和昆虫通道 DmNa(v)1 之间交换外部环，突出了参与各种毒素敏感性变化的通道区域。同时，对通道外部环的彻底突变阐明了与毒素相互作用的可能位点。rNa(v)1.2a 域 II 中的电压传感器模块的 S1-S2 和 S3-S4 外部环上的氨基酸取代对 beta 毒素 Css4（来自 Centruroides suffusus suffusus）的活性有显著影响，而 S1-S2 和 S3-S4 外部环上的电压传感器模块的取代在通道的活性影响了 alpha 毒素 Lqh2（来自 Leiurus quinquestriatus hebraeus）。使用钾通道的电压传感器模块作为模板对毒素-Na(v)相互作用进行 Rosetta 建模，揭示了 alpha 和 beta 毒素与通道相互作用的共同之处。Css4 与 rNa(v)1.2a 在域 II 中的 S1-S2 和 S3-S4 跨膜片段之间的缝隙中相互作用，而 Lqh2 与 rNa(v)1.2a 在域 IV 中的 S1-S2 和 S3-S4 跨膜片段之间的缝隙中相互作用。使用一系列 Lqh2 突变体对抗 rNa(v)1.2a 突变体的双突变循环分析和离解测定确定了 alpha 毒素在域 IV 中的门控模块的通道外表面上的对接方向。毒素与通道之间的另一个相互作用点尚未确定，可能涉及通道的 Pore-module 或 Gating-module 的残基。
