DataSheet1_Voltage-Gated Sodium Channel Modulation by a New Spider Toxin Ssp1a Isolated From an Australian Theraphosid.pdf

Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors.

鉴于电压门控钠离子通道（NaV）调节性蜘蛛毒素在阐明与治疗相关的NaV通道的功能、药理学和作用机制中的重要作用，本研究针对澳大利亚 Theraphosid 种类的毒液进行了筛选，以针对人类疼痛靶标hNaV1.7。通过采用测定指导的分级分离技术，我们从NaSpTx1家族中分离出一种由33个氨基酸组成的抑制性半胱氨酸结肽（ICK）肽（Ssp1a）。重组Ssp1a（rSsp1a）对神经元型hNaV亚型表现出显著的抑制活性，其活性顺序为hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1。rSsp1a能够抑制hNaV1.7、hNaV1.2和hNaV1.3，而不会显著改变其激活、失活或从失活恢复的电压依赖性。然而，rSsp1a在hNaV1.7上表现出电压依赖性的抑制，且rSsp1a结合的hNaV1.7在极端去极化时开放，这表明rSsp1a可能通过与hNaV1.7的电压感应域II（VSD II）相互作用，将通道锁定在其静息状态。核磁共振波谱揭示了Ssp1a的关键结构特征，包括一个两亲性表面，该表面具有疏水和带电的区域，对接研究表明这些区域构成了相互作用界面。本研究为未来结构-功能研究奠定了基础，以指导亚型选择性抑制剂的开发。