Department of Pharmacology, University of Washington, Seattle, WA 98195.
Department of Medicine, Division of General Internal Medicine, University of Washington, Seattle, WA 98195.
Proc Natl Acad Sci U S A. 2017 Apr 11;114(15):E3051-E3060. doi: 10.1073/pnas.1700761114. Epub 2017 Mar 27.
Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaAb mutants that capture tightly closed and open states at a resolution of 2.8-3.2 Å. Introduction of two humanizing mutations in the S6 segment (NaAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NaAb/1-226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaAb/1-226 as an open state that allows permeation of hydrated Na, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed-open-inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.
细菌电压门控钠离子通道（BacNavs）可作为其脊椎动物对应物的模型。BacNavs 包含保守的电压感应和孔形成结构域，但它们是由四个相同亚基组成的同源四聚体，而不是由四个同源结构域组成的拟四聚体。在这里，我们展示了两种 NaAb 突变体的结构，它们以 2.8-3.2 Å 的分辨率捕获了紧密关闭和开放状态。在 S6 片段中引入两个人类化突变（NaAb/FY：T206F 和 V213Y）会产生激活门的持续关闭形式，其中四个 S6 片段的细胞内末端紧密地拉在一起，完全阻止离子渗透。该结构还揭示了形成 C 末端结构域的四螺旋束的完整结构。相比之下，在 NaAb/1-226 中截断 C 末端的 40 个残基会捕获开放构象的激活门，从而揭示具有完整电压传感器的 BacNav 的开放状态。比较这些结构说明了激活门的全运动范围，从完全堵塞其孔口的关闭状态到打开时孔口约为 10 Å。分子动力学和自由能模拟证实了 NaAb/1-226 被指定为允许水合钠离子渗透的开放状态，这些结果还支持离子渗透控制的疏水性门控机制。这两种结构允许在单个电压门控钠离子通道中完成关闭-开放-失活构象循环，并深入了解钠离子通道阻断药物与状态相关的结合的结构基础。
