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

项目摘要/摘要 电压门控钠离子通道(NAV)是一种完整的膜蛋白，它选择性地将Na+离子传导到膜上 细胞膜。它们与心血管、神经和精神疾病有关，是 广泛使用的抗心律失常、抗惊厥药物的分子靶点。人类NaV1.5通道生成 心脏动作电位，并与危及生命的心律失常有关。海军的原子结构是 2011年首次从原核NAV通道获得，然后更多的真核NAV结构被解决 近几年被冷冻-EM发现，包括人类心脏的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 传感器和药理特征。最近，NAVAB的静止和激活构象 通道是通过结合功能依赖的交联和冷冻-EM来获得的，这为基础 进一步研究电压门控和药物调制机制的分子框架。我的项目 旨在揭示Navab和NaV1.5中选择性滤孔和电压传感器的动态行为 通道以及预留/阻断离子、门控电压和药物分子对通道的影响。我们会 实施尖端的单分子荧光共振能量转移(SmFRET)方法 实现这些拟议的研究。具体地说，我们将使用模型Navab和人类NaV1.5通道 为了(A)揭示钠选择性过滤器的构象灵活性和动力学，并阐明它如何 选择性地在K+和Ca~(2+)等阳离子上传导Na+；(B)确定选择性过滤器在慢速中的作用 失活，并了解利多卡因和氟卡胺等抗心律失常药物如何改变它们以抑制通道 功能；(C)显示电压传感器和通道的实时构象转变和动态 说明在Navab和NaV1.5通道中栅极是由电势直接驱动的，以阐明其机制 基础电压检测和选通。我们在纳瓦布海峡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。在重新提交中，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步，允许 美国将用荧光团标记人类NaV1.5通道用于smFRET研究。借助NaV1.5渠道，我们将 验证在Navab渠道上取得的关键发现，并揭示以下方面独有的动态属性 真核生物海军。我的研究将提供对离子选择性、电压 NAV通道的门控和药物调制，这将对其他通道和 运输商通过提供概念上的进步和新的方法。

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.