Structural dynamics of voltage-gated ion channels and their implications for ion permeation and drug modulation

项目总结/摘要 电压门控钠（Nav）通道是选择性地将Na+离子传导穿过膜的膜蛋白 细胞膜它们与心血管、神经和精神疾病有关， 广泛使用的抗惊厥药物的分子靶点。人类的Nav1.5通道 心脏动作电位，并与危及生命的心律失常有关。Navs的原子结构是 2011年首次从原核NavAb通道中获得，然后解决了更多的真核NavAb结构 近年来通过冷冻EM，包括人类心脏Nav1.5通道。原核生物和真核生物 NAV通道在结构上非常相似，包括它们的选择性过滤器、离子渗透孔、电压 传感器和药理学特征。最近，NavAb的静息和激活构象 通过功能依赖性交联和冷冻电镜相结合，获得了通道，这提供了基础 分子框架，以进一步研究电压门控和药物调节的机制。我的项目 旨在揭示NavAb和Nav1.5中选择性过滤孔和电压传感器的动力学行为 通道以及渗透/阻断离子、门控电压和药物分子对它们的影响。我们将 实施尖端的单分子荧光共振能量转移（smFRET）方法， 完成这些研究。具体而言，我们将使用模型NavAb和人Nav1.5通道 （a）揭示Na选择性过滤器的构象灵活性和动力学，并阐明它如何能够 选择性地传导Na+而不是阳离子如K+和Ca 2 +;（B）定义选择性过滤器在慢反应中的作用。 失活，并了解利多卡因和氟卡尼等抗肿瘤药物如何改变它们以抑制通道 （c）揭示电压传感器和通道的真实的时间构象转变和动力学 NavAb和Nav1.5通道中由电势直接驱动的门控，以阐明机制 潜在的电压感测和选通。我们已经在NavAb通道上获得了非常令人兴奋的初步数据， 这有力地证明了拟议研究的重要性和可行性。在重新提交时，我们进一步 通过建立非天然氨基酸掺入方法取得了关键技术进展， 我们用荧光团标记人Nav1.5通道用于smFRET研究。通过Nav1.5频道，我们将 验证NavAb通道上的关键发现，并揭示独特的动态特性， 真核Navs.我的研究将为离子选择性，电压 门控和药物调节Nav通道，这将对其他通道产生广泛的影响， 通过提供概念上的进步和新的方法来促进运输。

Project Summary/Abstract Voltage-gated sodium (Nav) channels are integral membrane proteins that selectively conduct Na+ ions across cell membranes. They are associated with cardiovascular, neurological, and psychiatric disorders and are the molecular targets of widely used antiarrhythmic, anticonvulsant drugs. The human Nav1.5 channel generates cardiac action potentials and is associated with life-threatening arrhythmias. The atomic structure of Navs was first obtained from a prokaryotic NavAb channel in 2011, and then more eukaryotic Nav structures were solved by cryo-EM in recent years, including the human cardiac Nav1.5 channel. Both the prokaryotic and eukaryotic Nav channels are very similar in structure, including their selectivity filters, ion permeation pores, voltage sensors, and pharmacological profiles. Most recently, the resting and activating conformations of NavAb channels were obtained by combining the function-dependent cross-linking and cryo-EM, which provided basic molecular frameworks to further investigate the mechanisms of voltage gating and drug modulation. My project aims to reveal dynamic behaviors of the selectivity filter pores and voltage sensors in NavAb and Nav1.5 channels and the effects of permeant/blocking ions, gating voltages, and drug molecules on them. We will implement the cutting-edge single molecule fluorescence resonance energy transfer (smFRET) approach to achieve these proposed studies. Specifically, we will use both the model NavAb and human Nav1.5 channels to (a) uncover the conformational flexibilities and dynamics of the Na selectivity filter and elucidate how it can selectively conduct Na+ over cations such as K+ and Ca2+; (b) define the roles of selectivity filters in slow inactivation, and understand how antiarrhythmic drugs like lidocaine and flecainide alter them to inhibit channel function; (c) reveal the real time conformational transitions and dynamics of the voltage sensor and channel gate in NavAb and Nav1.5 channels that is directly driven by the electrical potential to elucidate the mechanism underlying voltage sensing and gating. We have obtained very exciting preliminary data on the NavAb channel, which strongly justified the significance and feasibility of the proposed studies. In the resubmission, we further made the key technical advances by establishing the unnatural amino acid incorporation method, which allows us to label the human Nav1.5 channel with fluorophores for smFRET studies. With the Nav1.5 channel, we will validate the key findings made on the NavAb channel and reveal the dynamic properties that are unique for eukaryotic Navs. My studies will provide fundamental mechanistic insights into the ion selectivity, voltage gating, and drug modulation of Nav channels, which will have broad implications on other channels and transporters by providing both conceptual advances and novel methodologies.