Division of Biological and Biomedical Systems, School of Science and Engineering, University of Missouri-Kansas City, Kansas City, Missouri, USA.
Division of Biological and Biomedical Systems, School of Science and Engineering, University of Missouri-Kansas City, Kansas City, Missouri, USA.
J Biol Chem. 2023 Mar;299(3):102967. doi: 10.1016/j.jbc.2023.102967. Epub 2023 Feb 1.
High-resolution structures of voltage-gated sodium channels (Nav) were first obtained from a prokaryotic ortholog NavAb, which provided important mechanistic insights into Na selectivity and voltage gating. Unlike eukaryotic Navs, the NavAb channel is formed by four identical subunits, but its ion selectivity and pharmacological profiles are very similar to eukaryotic Navs. Recently, the structures of the NavAb voltage sensor at resting and activated states were obtained by cryo-EM, but its intermediate states and transition dynamics remain unclear. In the present work, we used liposome flux assays to show that purified NavAb proteins were functional to conduct both H and Na and were blocked by the local anesthetic lidocaine. Additionally, we examined the real-time conformational dynamics of the NavAb voltage sensor using single-molecule FRET. Our single-molecule FRET measurements on the tandem NavAb channel labeled with Cy3/5 FRET fluorophore pair revealed spontaneous transitions of the NavAb S4 segment among three conformational states, which fitted well with the kinetic model developed for the S4 segment of the human voltage-gated proton channel hHv1. Interestingly, even under strong activating voltage, the NavAb S4 segment seems to adopt a conformational distribution similar to that of the hHv1 S4 segment at a deep resting state. The conformational behaviors of the NavAb voltage sensor under different voltages need to be further examined to understand the mechanisms of voltage sensing and gating in the canonical voltage-gated ion channel superfamily.
电压门控钠离子通道（Nav）的高分辨率结构最初是从一个原核同源物 NavAb 中获得的，这为 Na 选择性和电压门控提供了重要的机制见解。与真核 Nav 不同，NavAb 通道由四个相同的亚基组成，但它的离子选择性和药理学特征与真核 Nav 非常相似。最近，通过 cryo-EM 获得了 NavAb 电压传感器在静息和激活状态下的结构，但它的中间状态和过渡动力学仍然不清楚。在本工作中，我们使用脂质体通量测定法表明，纯化的 NavAb 蛋白具有传导 H 和 Na 的功能，并且被局部麻醉剂利多卡因阻断。此外，我们使用单分子 FRET 研究了 NavAb 电压传感器的实时构象动力学。我们使用 Cy3/5 FRET 荧光团对串联 NavAb 通道进行的单分子 FRET 测量表明，NavAb S4 片段在三个构象状态之间自发转变，这与为人类电压门控质子通道 hHv1 的 S4 片段开发的动力学模型拟合良好。有趣的是，即使在强激活电压下，NavAb S4 片段似乎也采用了类似于 hHv1 S4 片段在深静息状态下的构象分布。需要进一步研究 NavAb 电压传感器在不同电压下的构象行为，以了解在典型电压门控离子通道超家族中电压感应和门控的机制。
