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 The nine subtypes of mammalian voltage-gated sodium (Nav) channels, Nav1.1 to Nav1.9, are responsible for the initiation and propagation of action potentials in specific excitable systems, among which Nav1.4 functions in skeletal muscle. Responding to membrane potential changes, Nav channels undergo sophisticated conformational shifts that lead to transitions between resting, activated, and inactivated states. Defects in Nav channels are associated with a variety of neurological, cardiovascular, muscular, and psychiatric disorders. In addition, Nav channels are targets for natural toxins and clinical therapeutics. Understanding the physiological and pathophysiological mechanisms of Nav channels requires knowing the structure of each conformational state. All eukaryotic Nav channels comprise a single polypeptide chain, the α subunit, that folds to four homologous repeats I to IV. Channel properties are modulated by one or two subtype-specific β subunits. Cryo–electron microscopy (cryo-EM) structures of two Nav channels, one from American cockroach and the other from electric eel, were resolved in two distinct conformations. However, the inability to record currents of either channel in heterologous systems prevented functional assignment of these structures. Structural elucidation of a functionally well-characterized Nav channel is required to establish a model for structure-function relationship studies. RATIONALE After extensive screening for expression systems, protein boundaries, chimeras, affinity tags, and combination with subtype-specific β subunits, we focused on human Nav1.4 in the presence of β1 subunit for cryo-EM analysis. The complex, which was transiently coexpressed in human embryonic kidney (HEK) 293F cells with BacMam viruses and purified through tandem affinity columns and size exclusion chromatography, was concentrated to ~0.5 mg/ml for cryo-EM sample preparation and data acquisition. RESULTS The cryo-EM structure of human Nav1.4-β1 complex was determined to 3.2-Å resolution. The extracellular and transmembrane domains, including the complete pore domain, all four voltage-sensing domains (VSDs), and the β1 subunit, were clearly resolved, enabling accurate model building (see the figure). The well-resolved Asp/Glu/Lys/Ala (DEKA) residues, which are responsible for specific Na+ permeation through the selectivity filter, exhibit identical conformations to those seen in the other two Nav structures. A glyco-diosgenin (GDN) molecule, the primary detergent used for protein purification and cryo-EM sample preparation, penetrates the intracellular gate of the pore domain, holding it open to a diameter of ~5.6 Å. The central cavity of the pore domain is filled with lipid-like densities, which traverse the side wall fenestrations. Voltage sensing involves four to six Arg/Lys residues on helix S4 of the VSD. This helix moves “up” (away from the cytoplasm) in response to changes of the membrane potential, and this opens the channel finally. All four VSDs display up conformations. The movement of the gating charge residues is facilitated by coordination to acidic and polar residues on S1 to S3. The improved resolution allows detailed analysis of the coordination. The fast inactivation Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV inserts into a hydrophobic cavity enclosed by the S6 and S4-S5 segments in repeats III and IV. Analysis of reported functional residues and disease mutations corroborates our recently proposed allosteric blocking mechanism for fast inactivation. CONCLUSION The structure provides important insight into the molecular basis for Na+ permeation, electromechanical coupling, asynchronous activation, and fast inactivation of the four repeats. It opens a new chapter for studying the structure-function relationships of Nav channels, affords an accurate template to map mutations associated with diseases such as myotonia and periodic paralysis hyperkalemic, and illuminates a path toward precise understanding and intervention with specific Nav channelopathies. Structure of the human Nav1.4-β1 complex. Two perpendicular views are shown. Left: Side view in ribbon cartoon. The VSDs are colored yellow, and the selectivity filter and supporting helices P1 and P2 are colored light cyan. The IFM motif is shown as spheres, and the III-IV linker is colored orange. The transmembrane segments in repeat IV are labeled. Right: Surface presentation for the bottom view to highlight the intracellular gate and the cavity that accommodates the IFM motif. The GDN molecule that penetrates the intracellular gate is shown as thin sticks. Voltage-gated sodium (Nav) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Nav channel has hampered mechanistic understanding. Here, we report the cryo–electron microscopy structure of the human Nav1.4-β1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the β1 subunit, providing insight into the molecular basis for Na+ permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.

中文翻译：

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与β1复合的人类电压门控钠通道Nav1.4的结构

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。引言 哺乳动物电压门控钠 (Nav) 通道的九种亚型，Nav1.1 到 Nav1.9，负责特定可兴奋系统中动作电位的启动和传播，其中 Nav1.4 在骨骼肌中起作用。响应膜电位变化，Nav 通道经历复杂的构象变化，导致静止、激活和失活状态之间的转换。导航通道的缺陷与多种神经、心血管、肌肉和精神疾病有关。此外，导航通道是天然毒素和临床治疗的目标。了解 Nav 通道的生理和病理生理机制需要了解每个构象状态的结构。所有真核 Nav 通道都包含一条多肽链，即 α 亚基，折叠成四个同源重复序列 I 至 IV。通道特性由一或两个亚型特异性 β 亚基调节。两个导航通道的冷冻电子显微镜 (cryo-EM) 结构，一个来自美国蟑螂，另一个来自电鳗，被解析为两种不同的构象。然而，无法记录异源系统中任一通道的电流阻止了这些结构的功能分配。需要对功能良好表征的导航通道进行结构阐明，以建立结构-功能关系研究模型。基本原理在对表达系统、蛋白质边界、嵌合体、亲和标签以及与亚型特异性 β 亚基的组合进行广泛筛选后，我们专注于在存在 β1 亚基的情况下对人类 Nav1.4 进行冷冻电镜分析。该复合物在人胚胎肾 (HEK) 293F 细胞中与 BacMam 病毒瞬时共表达，并通过串联亲和柱和尺寸排阻色谱纯化，浓缩至约 0.5 mg/ml，用于冷冻电镜样品制备和数据采集。结果 人类 Nav1.4-β1 复合物的冷冻电镜结构确定为 3.2 Å 分辨率。细胞外和跨膜结构域，包括完整的孔结构域、所有四个电压感应域 (VSD) 和 β1 亚基，都得到了清晰的解析，从而实现了准确的模型构建（见图）。解析良好的 Asp/Glu/Lys/Ala (DEKA) 残基负责特定 Na+ 通过选择性过滤器的渗透，表现出与其他两个 Nav 结构中所见构象相同的构象。糖苷元 (GDN) 分子是用于蛋白质纯化和冷冻 EM 样品制备的主要洗涤剂，它穿透孔域的细胞内门，使其直径保持在 ~5.6 Å。孔域的中央空腔充满脂质样密度，穿过侧壁开窗。电压感应涉及 VSD 螺旋 S4 上的四到六个 Arg/Lys 残基。该螺旋响应膜电位的变化“向上”（远离细胞质）移动，这最终打开了通道。所有四个 VSD 都显示了构象。通过与 S1 到 S3 上的酸性和极性残基的配位促进门控电荷残基的移动。改进的分辨率允许对协调进行详细分析。重复 III 和 IV 之间的短接头上的快速失活 Ile/Phe/Met (IFM) 基序插入到由重复 III 和 IV 中的 S6 和 S4-S5 片段包围的疏水腔中。对报告的功能残基和疾病突变的分析证实了我们最近提出的用于快速灭活的变构阻断机制。结论 该结构为 Na+ 渗透、机电耦合、异步激活和四个重复的快速失活的分子基础提供了重要的见解。开启了Nav通道结构-功能关系研究的新篇章，提供了一个准确的模板来绘制与肌强直和周期性麻痹高钾血症等疾病相关的突变，并为精确理解和干预特定的 Nav 通道病指明了道路。人类 Nav1.4-β1 复合物的结构。显示了两个垂直视图。左：丝带卡通侧视图。VSD 为黄色，选择性过滤器和支撑螺旋​​ P1 和 P2 为浅青色。IFM 基序显示为球体，III-IV 接头为橙色。重复 IV 中的跨膜片段被标记。右图：底视图的表面呈现，以突出显示细胞内门和容纳 IFM 基序的腔。穿透细胞内门的 GDN 分子显示为细棒。电压门控钠 (Nav) 通道，它们负责产生动作电位，与许多人类疾病有关。尽管进行了数十年的严格表征，但缺乏任何人类导航通道的结构阻碍了机械理解。在这里，我们报告了人类 Nav1.4-β1 复合物的冷冻电子显微镜结构，分辨率为 3.2 Å。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。与许多人类疾病有关。尽管进行了数十年的严格表征，但缺乏任何人类导航通道的结构阻碍了机械理解。在这里，我们报告了人类 Nav1.4-β1 复合物的冷冻电子显微镜结构，分辨率为 3.2 Å。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。与许多人类疾病有关。尽管进行了数十年的严格表征，但缺乏任何人类导航通道的结构阻碍了机械理解。在这里，我们报告了人类 Nav1.4-β1 复合物的冷冻电子显微镜结构，分辨率为 3.2 Å。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。缺乏任何人类导航通道的结构阻碍了机械理解。在这里，我们报告了人类 Nav1.4-β1 复合物的冷冻电子显微镜结构，分辨率为 3.2 Å。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。缺乏任何人类导航通道的结构阻碍了机械理解。在这里，我们报告了人类 Nav1.4-β1 复合物的冷冻电子显微镜结构，分辨率为 3.2 Å。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。2-Å 分辨率。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。2-Å 分辨率。对孔域、电压感应域和 β1 亚基进行了准确的模型构建，提供了对 Na+ 渗透和四个重复的动力学不对称性的分子基础的深入了解。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。报告的功能残基和疾病突变的结构分析证实了 Nav 通道快速失活的变构阻断机制。该结构为导航通道的机械研究和导航通道病的药物发现提供了一条途径。