Uncovering the Mechanism of Potassium Channel Folding and Assembly with Implications for the Molecular Basis of Cardiac Arrhythmia

揭示钾通道折叠和组装的机制对心律失常的分子基础的影响

• 批准号: 10389217
• 负责人: Andrew Vincent Molina
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389217
• 关键词: Affect; Agreement; Arrhythmia; Behavior; Biochemical; Biogenesis; Biological; Biological Models; Biophysics; Brugada syndrome; Cardiac; Cardiac Myocytes; Cardiac health; Cell physiology; Cells; Complement; Disease; Engineering; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Future; Grain; Human; Hydrogen; Image; In Vitro; Ion Channel; Ion Transport; Ions; Kinetics; Label; Lead; Life; Light; Lipid Bilayers; Long QT Syndrome; Mass Spectrum Analysis; Membrane; Membrane Lipids; Membrane Proteins; Methods; Microscopy; Missense Mutation; Modeling; Molecular; Mutation; Nature; Pathogenesis; Pathologic; Pathway interactions; Physiologic pulse; Potassium Channel; Process; Proteins; Publishing; Regulation; Research; Resolution; Role; Rotation; Scanning Probe Microscopy; Short QT syndrome; Spectrum Analysis; Speed; Syndrome; Techniques; Temperature; Variant; Ventricular Fibrillation; Vesicle; Water; Work; biophysical techniques; disease-causing mutation; disulfide bond; heart function; heart rhythm; in vivo; innovation; insight; monomer; novel; physical property; potassium ion; prevent; protein folding; reconstitution; simulation

Project Summary/Abstract Potassium channels are membrane proteins critical for the electrochemical regulation and function of cardiac cells. Many diseases are associated with mutations in human potassium channels, including Long-QT Syndrome, Short-QT Syndrome, Brugada Syndrome, Lev-Lenegre Syndrome, and Idiopathic Ventricular Fibrillation. The molecular basis of these diseases remains poorly understood, and many arrhythmia-associated mutations may directly disrupt protein folding. Therefore, it is essential to study the mechanism and biophysical determinants of potassium channel folding to understand how these mutations may result in arrhythmia. Preliminary work is presented here on the in vitro folding of the KcsA transmembrane pore domain, a robust model system for human potassium channels such as hERG and Kv1.2. This work suggests that KcsA rapidly inserts as monomers into a protein-dense region within the lipid membrane, and tetramerization kinetics are protein concentration-independent, implying a unimolecular rate-limiting step despite the tetrameric nature of the channel. These observations raise the following questions: What is the role of the protein-dense region in potassium channel folding? What are the structural events in potassium channel folding, specifically regarding the rate-limiting step? Lastly, and most relevant to cardiac health, how might missense mutations of the pore helix, such as A614V, L615V, and T623I of hERG, disrupt folding and lead to arrhythmia? The proposed work will investigate the protein-dense region using super-resolution light and scanning-probe microscopy in reconstituted membranes and live HL-1 cardiomyocytes to evaluate the hypothesis that the protein-dense region functions to quickly concentrate channel monomers in the membrane and thus increase the speed and efficiency of folding. To determine the structural events in channel folding, we will use a novel hydrogen-exchange mass spectrometry (HXMS) technique alongside other biophysical methods to evaluate the hypothesis that folding must occur by one of two possible mechanisms: (1) a “native assembly model” in which four natively-folded channel monomers assemble in a single, concerted step, or (2) a “keystone model” in which the transmembrane helices of each monomer initially tetramerize into a transmembrane bundle, and then the pore helix and selectivity filters insert into and stabilize the channel like the keystone of an arch. Pulse-labeling and native state HXMS will probe the folding dynamics and stability, respectively, of channel variants associated with Long-QT Syndrome to evaluate the hypothesis that pore helix missense mutations can cause disease by preventing proper pore helix folding. These approaches will be complemented by computational coarse-grained and all- atom techniques, including a novel “committor” analysis method to study the reactive flux between metastable folded and unfolded potassium channel states. The proposed work is high impact: It uses innovative and interdisciplinary techniques such as HXMS to uncover the mechanism of potassium channel folding and its implications for cardiac arrhythmia. These insights will inform future studies of membrane protein folding biophysics as well as the pathogenesis of heart rhythm disorders.

项目概要/摘要 钾通道是对心肌细胞的电化学调节和功能至关重要的膜蛋白。 许多疾病都与人类钾通道突变有关，包括长 QT 综合征、短 QT 综合征 综合征、Brugada 综合征、Lev-Lenegre 综合征和特发性心室颤动的分子基础。 这些疾病仍然知之甚少，许多与心律失常相关的突变可能直接破坏蛋白质折叠。 因此，有必要研究钾通道折叠的机制和生物物理决定因素，以了解钾通道折叠的机制。 这些突变如何导致心律失常。 本文介绍了 KcsA 跨膜孔域体外折叠的初步工作，这是一个稳健的模型 hERG 和 Kv1.2 等人钾通道系统这项工作表明 KcsA 作为单体快速插入。 进入脂膜内的蛋白质密集区域，四聚化动力学与蛋白质浓度无关， 尽管通道具有四聚体性质，但这意味着单分子限速步骤。 以下问题： 蛋白质密集区在钾通道折叠中的作用是什么？ 钾通道折叠中的事件，特别是关于限速步骤？最后，也是与心脏健康最相关的， 孔螺旋的错义突变（例如 hERG 的 A614V、L615V 和 T623I）如何破坏折叠并导致 心律失常？拟议的工作将使用超分辨率光和扫描探针研究蛋白质密集区域 在重建膜和活 HL-1 心肌细胞中进行显微镜检查，以评估蛋白质密集的假设 该区域的作用是快速浓缩膜中的通道单体，从而提高通道单体的速度和效率 为了确定通道折叠中的结构事件，我们将使用一种新型的氢交换质谱法。 （HXMS）技术与其他生物物理方法一起评估折叠必须通过以下两种方式之一发生的假设 可能的机制：（1）“天然组装模型”，其中四个天然折叠通道单体组装在一个单一的、 协调一致的步骤，或（2）“基石模型”，其中每个单体的跨膜螺旋最初四聚成 跨膜束，然后孔螺旋和选择性过滤器像梯形石一样插入并稳定通道 脉冲标记和天然状态 HXMS 将分别探测通道的折叠动力学和稳定性。 与长 QT 综合征相关的变异，用于评估孔螺旋错义突变可导致疾病的假设 通过防止适当的孔螺旋折叠，这些方法将得到计算粗粒度和全结构的补充。 原子技术，包括一种新颖的“提交者”分析方法，用于研究亚稳态折叠和 钾通道展开状态。 拟议的工作具有很高的影响力：它使用 HXMS 等创新和跨学科技术来揭示 钾通道折叠的机制及其对心律失常的影响这些见解将为未来提供信息。 研究膜蛋白折叠生物物理学以及心律失常的发病机制。