Structures of voltage-gated sodium channels In “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Nav) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Nav channel, and Shen et al. report the structures of an insect Nav channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery. Science, this issue p. eaau2486, p. eaau2596 Structures provide insight into how voltage-gated sodium channels function and how they can be inhibited. INTRODUCTION Almost all venoms contain toxins that modulate the activity of voltage-gated sodium (Nav) channels in order to incapacitate prey or predators. The single-chain eukaryotic Nav channels comprise four homologous repeats. The central pore domain is constituted by the carboxyl-terminal segments from all four repeats, and each repeat also has a voltage-sensing domain (VSD). Toxins are broadly divided into two categories—pore blockers that physically occlude the channel pore and gating modifiers that alter channel gating by interfering with the VSDs. Whereas small-molecule neurotoxins such as tetrodotoxin (TTX) and saxitoxin (STX) function as pore blockers, most peptidic Nav channel toxins are gating modifiers that trap the channel in a particular stage of the gating cycle through interactions with one or more VSDs. In neither case is the structural basis of channel modulation fully understood. RATIONALE Dc1a is a peptidic gating modifier toxin (GMT) from venom of the desert bush spider Diguetia canities that specifically binds to VSDII of insect Nav channels to promote channel opening. We showed through biochemical analysis that Dc1a interacts with NavPaS, a Nav channel from the American cockroach Periplaneta americana, for which a cryo–electron microscopy (cryo-EM) structure was recently determined at 3.8-Å resolution. We therefore sought to solve the structure of the complex between NavPaS and Dc1a. As Dc1a occupies a distinctly different channel binding site to pore blockers, we also attempted to supplement the complex with TTX or STX to obtain structures of the ternary complexes. RESULTS The cryo-EM structure of NavPaS-Dc1a was determined to an overall resolution of 2.8 Å in the presence of 300 mM NaCl, whereas those of NavPaS-Dc1a-TTX and NavPaS-Dc1a-STX were resolved at 2.6 Å and 3.2 Å, respectively, in the presence of 150 mM NaCl. VSDII constitutes the primary docking site for Dc1a, which undergoes considerable structural rearrangement upon binding to the channel. The toxin inserts into the cleft between VSDII and the pore region, making intimate contacts with both domains. The network of intermolecular interactions seen in the cryo-EM structure was validated through examination of the effect of toxin and channel mutations using the orthologous NavBg channel from the German cockroach Blattella germanica. Four residues, Asp/Glu/Lys/Ala (DEKA), at a corresponding locus in the selectivity filter (SF) of each repeat confer Na+ selectivity. A Na+ ion was observed in the same position in the structures of NavPaS-Dc1a and NavPaS-Dc1a-TTX, coordinated by the Asp and Glu residues in the DEKA motif of the SF, and an invariant Glu on the P2 helix in repeat II, a helix in the entryway to the SF on the extracellular side. Both TTX and STX form extensive electrostatic interactions with residues in the outer electronegative ring that attracts cations into the SF and Asp and Glu in the DEKA motif, completely blocking access of Na+ ions to the SF. CONCLUSION The structure of the NavPaS-Dc1a complex suggests that the network of interactions between Nav channels and GMTs is more complex than previously anticipated. Therefore, caution has to be applied when using isolated Nav channel VSDs for drug discovery or for understanding the molecular basis of GMT action. The current structures elucidate the molecular basis for the insect selectivity of Dc1a and the subtype-specific binding of TTX or STX to Nav channels. Unambiguous structural elucidation of the bound TTX and STX, whose molecular weights are both around 300 Da, showcases the power of cryo-EM and its potential for structure-aided drug discovery. Structural basis for specific binding of GMT Dc1a and guanidinium pore blockers TTX and STX by NavPaS. (A) Dc1a inserts into the extracellular cavity between VSDII and the pore elements of repeat III. (B) Molecular mechanism for pore blockade by TTX and STX. Top: The carboxylate groups of Asp (D) and Glu (E) residues in the DEKA motif and an invariant Glu on P2II together constitute a potential Na+ binding site (designated the DEE site). Bottom: TTX and STX block access of Na+ to the DEE site from the extracellular side. A semitransparent presentation of the electrostatic surface potential of the entrance to the SF viewed from the extracellular side is shown. CTD, C-terminal domain; R, Arg; L, Leu; Y, Tyr; K, Lys. Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories—pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Nav channel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDII and the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+ access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Nav channel drugs.

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动物毒素调节电压门控钠通道的结构基础

电压门控钠通道的结构在“可兴奋”细胞中，如神经元和肌肉细胞，电位差用于跨细胞膜传输信号。这种差异是通过打开或关闭细胞膜中的离子通道来调节的。例如，人类电压门控钠 (Nav) 通道的突变与慢性疼痛、癫痫和心律失常等疾病有关。潘等人。报告了人类导航通道的高分辨率结构，Shen 等人。报告了与导致人类河豚和贝类中毒的毒素结合的昆虫导航通道的结构。这些结构共同揭示了钠离子渗透的分子基础，并为基于结构的药物发现提供了途径。科学，这个问题 p。eaau2486，第。eaau2596 结构可以深入了解电压门控钠通道的功能以及如何抑制它们。介绍 几乎所有的毒液都含有毒素，可以调节电压门控钠 (Nav) 通道的活性，从而使猎物或捕食者失去能力。单链真核导航通道包含四个同源重复。中心孔域由来自所有四个重复的羧基末端片段构成，每个重复也有一个电压感应域（VSD）。毒素大致分为两类——物理堵塞通道孔的孔阻滞剂和通过干扰 VSD 改变通道门控的门控调节剂。河豚毒素 (TTX) 和石房蛤毒素 (STX) 等小分子神经毒素可作为毛孔阻滞剂，大多数肽类 Nav 通道毒素是门控修饰剂，通过与一个或多个 VSD 的相互作用在门控循环的特定阶段捕获通道。在这两种情况下，都没有完全理解信道调制的结构基础。基本原理 Dc1a 是一种来自沙漠灌木蜘蛛 Diguetia canities 毒液的肽门控修饰剂毒素 (GMT)，它与昆虫导航通道的 VSDII 特异性结合以促进通道开放。我们通过生化分析表明，Dc1a 与 NavPaS 相互作用，NavPaS 是来自美国大蠊美洲大蠊的导航通道，最近在 3.8 Å 分辨率下确定了低温电子显微镜 (cryo-EM) 结构。因此，我们试图解决 NavPaS 和 Dc1a 之间复合物的结构。由于 Dc1a 占据与孔阻滞剂明显不同的通道结合位点，我们还尝试用 TTX 或 STX 补充复合物以获得三元复合物的结构。结果 在 300 mM NaCl 存在下，NavPaS-Dc1a 的冷冻电镜结构确定为 2.8 Å，而 NavPaS-Dc1a-TTX 和 NavPaS-Dc1a-STX 的总分辨率为 2.6 Å 和 3.2 Å，分别在 150 mM NaCl 存在下。VSDII 构成 Dc1a 的主要停靠位点，Dc1a 在与通道结合后会发生相当大的结构重排。毒素插入 VSDII 和孔隙区域之间的裂隙中，与两个结构域密切接触。通过使用德国蟑螂德国小蠊的直系同源 NavBg 通道检查毒素和通道突变的影响，验证了在冷冻电镜结构中看到的分子间相互作用网络。四个残基，Asp/Glu/Lys/Ala (DEKA)，在每个重复的选择性过滤器 (SF) 的相应位点上，赋予 Na+ 选择性。在 NavPaS-Dc1a 和 NavPaS-Dc1a-TTX 结构中的相同位置观察到 Na+ 离子，由 SF 的 DEKA 基序中的 Asp 和 Glu 残基协调，以及重复 II 中 P2 螺旋上的不变 Glu，细胞外 SF 入口通道中的螺旋。TTX 和 STX 都与外带负电环中的残基形成广泛的静电相互作用，将阳离子吸引到 SF 中以及 DEKA 基序中的 Asp 和 Glu，完全阻止 Na+ 离子进入 SF。结论 NavPaS-Dc1a 复合物的结构表明 Nav 通道和 GMT 之间的交互网络比以前预期的更复杂。所以，在使用孤立的导航通道 VSD 进行药物发现或了解 GMT 作用的分子基础时，必须谨慎。目前的结构阐明了 Dc1a 的昆虫选择性和 TTX 或 STX 与 Nav 通道的亚型特异性结合的分子基础。结合 TTX 和 STX 的明确结构解析，其分子量均在 300 Da 左右，展示了冷冻电镜的力量及其在结构辅助药物发现方面的潜力。NavPaS 特异性结合 GMT Dc1a 和胍孔阻滞剂 TTX 和 STX 的结构基础。(A) Dc1a 插入到 VSDII 和重复 III 的孔元件之间的细胞外腔中。(B) TTX 和 STX 阻塞孔的分子机制。最佳：DEKA 基序中的 Asp (D) 和 Glu (E) 残基的羧基和 P2II 上的不变 Glu 共同构成潜在的 Na+ 结合位点（指定为 DEE 位点）。底部：TTX 和 STX 阻止 Na+ 从细胞外侧进入 DEE 位点。显示了从细胞外侧观察到的 SF 入口的静电表面电位的半透明表示。CTD，C端域；R，精氨酸；L，列伊；Y，提尔；K，赖斯。调节电压门控钠 (Nav) 通道活性的动物毒素大致分为两类——孔阻滞剂和门控调节剂。毛孔阻滞剂河豚毒素 (TTX) 和石房蛤毒素 (STX) 分别是导致人类河豚和贝类中毒的原因。在这里，我们展示了昆虫 Nav 通道 NavPaS 的结构，该结构与 2 处的门控修饰符毒素 Dc1a 结合。8 埃分辨率和存在 TTX 或 STX 的情况下，分辨率分别为 2.6-Å 和 3.2-Å。Dc1a 插入到 VSDII 和 NavPaS 孔之间的裂缝中，与两个域建立关键联系。结合 TTX 或 STX 的结构揭示了这些胍毒素从细胞外侧特异性阻断 Na+ 进入选择性过滤器的分子细节。这些结构阐明了基于结构的 Nav 通道药物开发。结合 TTX 或 STX 的结构揭示了这些胍毒素从细胞外侧特异性阻断 Na+ 进入选择性过滤器的分子细节。这些结构阐明了基于结构的 Nav 通道药物开发。结合 TTX 或 STX 的结构揭示了这些胍毒素从细胞外侧特异性阻断 Na+ 进入选择性过滤器的分子细节。这些结构阐明了基于结构的 Nav 通道药物开发。