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

电压门控离子通道的结构动力学及其对离子渗透和药物调节的影响

• 批准号: 10583283
• 负责人: SHIZHEN WANG
• 金额: $ 32.87万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583283
• 关键词: Action Potentials; Address; Anti-Arrhythmia Agents; Anticonvulsants; Arrhythmia; Behavior; Binding; Biological Assay; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cations; Cell membrane; Cells; Charge; Chemistry; Complex; Coupling; Cryoelectron Microscopy; Cysteine; Data; Drug Modulation; Drug Regulations; Electrophysiology (science); Environment; Exhibits; Flecainide; Goals; Helix-Turn-Helix Motifs; Human; Integral Membrane Protein; Ion Channel Gating; Ions; Label; Lidocaine; Life; Liposomes; Maleimides; Membrane; Membrane Proteins; Mental disorders; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Molecular Target; Pharmaceutical Preparations; Property; Protons; Regulation; Resolution; Rest; Role; Sodium; Sodium Channel; Solid; Specificity; Structure; Techniques; Time; Tissues; Visualization; conformational conversion; crosslink; drug modification; electrical potential; flexibility; fluorescence imaging; fluorophore; functional status; insight; method development; nervous system disorder; next generation; novel; patch clamp; pharmacologic; sensor; single molecule; single-molecule FRET; unnatural amino acids; voltage

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.

项目摘要/摘要 电压门控钠（NAV）通道是整体膜蛋白，在跨越跨越Na+离子 细胞膜。它们与心血管，神经系统和精神疾病有关，是 广泛使用的抗心律失常，抗惊厥药的分子靶标。人类NAV1.5频道生成 心脏作用潜力，与威胁生命的心律失常有关。 NAV的原子结构是 首先是从原核Navab通道获得的，然后解决了更多的真核NAV结构 近年来，由Cryo-Em撰写，包括人类心脏NAV1.5通道。原核生物和真核 NAV通道的结构非常相似，包括其选择性过滤器，离子渗透孔，电压 传感器和药理学概况。最近，Navab的休息和激活构象 通过组合功能依赖性的交联和冷冻EM获得通道，该通道提供了基本 分子框架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Navab和Nav1.5中选择性过滤孔和电压传感器的动态行为 通道和渗透性/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传递（SMFRET）方法 实现这些建议的研究。具体来说，我们将同时使用Navab和Human Nav1.5频道 （a）揭示Na选择性过滤的构象灵活性和动力学，并阐明如何 选择性地对K+和Ca2+等阳离子进行Na+； （b）在慢速中定义选择性过滤器的作用 失活，并了解利多卡因和氟卡因等抗心律失常药物如何改变它们以抑制通道 功能; （c）揭示电压传感器和通道的实时构象过渡和动力学 Navab和Nav1.5通道中的门直接由电势驱动以阐明机制 基础电压传感和门控。我们在Navab频道上获得了非常令人兴奋的初步数据， 这很大程度上证明了拟议的研究的重要性和可行性。在重新提交中，我们进一步 通过建立非自然氨基酸掺入方法来取得关键的技术进步，该方法允许 我们将人NAV1.5通道标记为荧光团进行SMFRET研究。使用NAV1.5频道，我们将 验证在Navab通道上提出的关键发现，并揭示独特的动态属性 真核N​​AVS。我的研究将为离子选择性，电压提供基本的机械见解 NAV通道的门控和药物调节，这将对其他渠道具有广泛的影响， 通过提供概念进步和新颖的方法来转运者。