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

项目摘要/摘要 钾通道是一种膜蛋白，对心肌细胞的电化学调节和功能至关重要。 许多疾病与人类钾通道的突变有关，包括长QT综合征、短QT 综合征、Brugada综合征、Lev-Lenegre综合征和特发性室颤。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扰乱蛋白质折叠。 因此，研究钾通道折叠的机制和生物物理决定因素对于理解钾通道折叠是非常必要的。 这些突变是如何导致心律失常的。 本文介绍了KCSA跨膜孔域的体外折叠的初步工作，这是一个健壮的模型。 人的钾通道系统，如HERG和Kv1.2。这项工作表明KCSA以单体的形式快速插入 进入脂膜内的蛋白质致密区域，四聚动力学不依赖于蛋白质浓度， 意味着单分子限速步骤，尽管通道的四聚体性质。这些观察结果提高了 以下问题：蛋白密集区在钾通道折叠中的作用是什么？什么是结构上的 钾通道折叠中的事件，特别是关于速率限制步骤？最后，也是与心脏健康最相关的， 毛孔螺旋的错义突变，如HERG的A614V、L615V和T623I，如何破坏折叠并导致 心律不齐?拟议中的工作将使用超分辨率光和扫描探针来研究蛋白质致密区域 在重组膜和活的HL-1心肌细胞中进行显微镜观察以评估蛋白质致密的假设 区域的作用是快速将通道单体集中在膜中，从而提高速度和效率 折叠。为了确定通道折叠中的结构事件，我们将使用一种新的氢交换质谱学 (HXMS)技术和其他生物物理方法一起评估折叠必须发生在两个中的一个的假设 可能的机制：(1)其中四个自然折叠的通道单体在单个， 协调步骤，或(2)“基石模型”，在该模型中每个单体的跨膜螺旋最初四聚为 跨膜束，然后孔螺旋和选择性过滤器插入并稳定通道，就像拱心石一样 一座拱门。脉冲标记和自然状态HXMS将分别探测通道的折叠动力学和稳定性 与长QT综合征相关的变异评估毛孔螺旋错义突变可致病的假说 通过防止适当的毛孔螺旋折叠。这些方法将由计算粗粒度和所有- 原子技术，包括一种新的“委托人”分析方法，以研究亚稳折叠和亚稳折叠之间的反应通量 未折叠的钾通道状态。 拟议的工作具有很高的影响力：它使用创新和跨学科的技术，如HXMS来揭示 钾通道折叠机制及其在心律失常中的意义。这些见解将为未来 膜蛋白折叠生物物理学以及心律失常的发病机制研究。

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.