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

项目总结/摘要 钾通道是心肌细胞膜上的一种蛋白质，对心肌细胞的电化学调节和功能起着重要作用。 许多疾病都与人体钾离子通道的突变有关，包括长QT综合征、短QT综合征、 综合征、Brugada综合征、Lev-Lenegre综合征和特发性室颤。的分子基础 这些疾病仍然知之甚少，许多与疟疾相关的突变可能直接破坏蛋白质折叠。 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的折叠是非常必要的。 这些突变是如何导致心律失常的 初步工作是在体外折叠的KcsA跨膜孔结构域，一个强大的模型 人类钾通道系统，如hERG和Kv1.2。这项工作表明，KcsA作为单体快速插入 进入脂质膜内的蛋白质密集区，并且四聚动力学不依赖于蛋白质浓度， 这意味着单分子速率限制步骤，而不管通道的四聚体性质。这些观察提出了 以下问题：蛋白质致密区在钾通道折叠中的作用是什么？什么是结构 钾通道折叠的事件，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，如hERG的A614 V、L 615 V和T623 I，如何破坏折叠并导致 心律不齐本论文将利用超分辨光和扫描探针对蛋白质密集区进行研究 在重组膜和活的HL-1心肌细胞中进行显微镜检查，以评估蛋白质致密的 该区域的功能是快速浓缩膜中的通道单体，从而提高聚合的速度和效率。 折页.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法 （HXMS）技术与其他生物物理方法一起评估折叠必须通过两种方式之一发生的假设。 可能的机制：（1）“天然组装模型”其中四个天然折叠的通道单体组装成单个， 协同步骤，或（2）“梯形模型”，其中每个单体的跨膜螺旋最初四聚成一个 然后，孔螺旋和选择性过滤器插入并稳定通道，如梯形 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，以评估孔螺旋错义突变可导致疾病的假设 通过阻止正确的孔螺旋折叠。这些方法将通过计算粗粒度和所有- 原子技术，包括一种新的“提交人”分析方法，以研究亚稳折叠和 未折叠钾通道状态。 拟议的工作是高影响力：它使用创新和跨学科的技术，如HXMS，以揭示 钾通道折叠的机制及其在心律失常中的意义。这些见解将告知未来 研究膜蛋白折叠生物物理学以及心脏节律紊乱的发病机制。

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.