Calmodulin Regulation of KCNQ1 Potassium Channels

KCNQ1 钾通道的钙调蛋白调节

• 批准号: 9907807
• 负责人: Po wei Kang
• 金额: $ 5.05万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907807
• 关键词: Action Potentials; Acute; Address; Affect; Affinity; Arrhythmia; Binding; Binding Sites; Biological Assay; Calmodulin; Cardiac; Cardiac Myocytes; Chimera organism; Complex; Cryoelectron Microscopy; Cytoplasm; Cytoplasmic Tail; Data; Disabled Persons; Drug Targeting; Electrodes; Electrophysiology (science); Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorometry; Functional disorder; Goals; Heart; Inherited; Ion Channel; Kinetics; Lead; Ligands; Lobe; Long QT Syndrome; Methods; Modeling; Molecular; Mutagenesis; Mutation; Optics; Patients; Pharmacology; Phosphatidylinositol 4,5-Diphosphate; Potassium; Potassium Channel; Process; Protein Engineering; Protein Isoforms; Regulation; Romano-Ward Syndrome; Stimulus; Structure; Techniques; Testing; Therapeutic; Transmembrane Domain; Voltage-Gated Potassium Channel; base; biophysical techniques; insight; mutant; novel; patch clamp; precision medicine; response; sensor; sudden cardiac death; tool; trafficking; voltage; voltage clamp

PROJECT SUMMARY In the heart, KCNQ1 voltage-gated potassium channels form the alpha-subunits for the slow delayed rectifier potassium current, a repolarizing current critical for cardiac action potential termination. Calmodulin (CaM) is an obligatory KCNQ1 auxiliary subunit which binds the KCNQ1 cytoplasmic domain and regulates normal KCNQ1 trafficking and function. Inherited mutations in KCNQ1 (>600) and CaM lead to congenital long QT syndrome (LQTS), which predisposes patients to arrhythmias and sudden cardiac death. Little is known regarding the specific KCNQ1 mutations or the molecular mechanism underlying CaM dysregulation of KCNQ1 to cause LQTS. Conversely, because CaM binds multiple ion channel targets in cardiomyocytes, it is unclear whether CaM mutations cause LQTS by inducing KCNQ1 dysfunction. This study’s overall goal is to elucidate mechanisms of CaM regulation of KCNQ1 and identify arrhythmogenic mutations in KCNQ1 and CaM which cause LQTS specifically through CaM dysregulation. Until recently, the lack of KCNQ1 structural and gating mechanism insights has hindered mechanistic studies of CaM regulation of KCNQ1. However, recent breakthroughs in both functional and structural studies of KCNQ1 revealed novel findings which may overcome this barrier. This proposal is motivated by three novel CaM regulatory hypotheses derived from these studies: (1) CaM regulates KCNQ1 through simultaneous interactions with the KCNQ1 cytoplasmic and transmembrane domains, (2) CaM exerts selective regulation on distinct KCNQ1 open states, and (3) CaM regulation requires concurrent ATP binding. Presently, there is little functional data to support these hypotheses as genuine gating mechanisms. This proposal plans to investigate these novel CaM regulatory hypotheses with three aims. Aim 1 will characterize the functional impact of disrupting CaM simultaneous interactions with the transmembrane and cytoplasmic domains with systematic mutagenesis, electrophysiological recordings, and optical assays. Aim 2 will utilize a biophysical approach to probe the mechanistic hypothesis that CaM alters KCNQ1 current through selective regulation of distinct KCNQ1 open states with mutagenesis and protein engineering methods. Aim 3 will expand the scope to multi-ligand KCNQ1 regulation. Aim 3 will answer whether CaM regulation of KCNQ1 requires concurrent ATP binding to the KCNQ1 cytoplasmic domain with combined electrophysiology and fluorescence techniques. Results from this study will functionally establish novel mechanisms of CaM regulation of KCNQ1, identify which and how mutations in KCNQ1 and CaM lead to LQTS due to CaM dysregulation. These findings will pave the way for a precision medicine approach to congenital LQTS and may reveal therapeutic approaches against LQTS through targeting these novel CaM regulatory mechanisms.

项目概要 在心脏中，KCNQ1 电压门控钾通道形成缓慢延迟整流器的 α 亚基 钾电流，一种对心脏动作电位终止至关重要的复极电流。 Calmodulin (CaM) is an 必需的 KCNQ1 辅助亚基，结合 KCNQ1 胞质结构域并调节正常的 KCNQ1 trafficking and function. KCNQ1 (>600) 和 CaM 的遗传性突变导致先天性长 QT 综合征 （LQTS），使患者容易出现心律失常和心源性猝死。 Little is known regarding the 特定的 KCNQ1 突变或 KCNQ1 CaM 失调的分子机制导致 LQTS.相反，由于 CaM 结合心肌细胞中的多个离子通道靶标，因此尚不清楚是否 CaM 突变通过诱导 KCNQ1 功能障碍导致 LQTS。 This study’s overall goal is to elucidate CaM 对 KCNQ1 的调节机制，并鉴定 KCNQ1 和 CaM 中的致心律失常突变，这些突变 特别是通过 CaM 失调引起 LQTS。直到最近，KCNQ1 结构和门控的缺失 机制的见解阻碍了 CaM 对 KCNQ1 调节的机制研究。然而，最近 KCNQ1 功能和结构研究的突破揭示了可能克服的新发现 this barrier.该提案的动机是源自这些研究的三个新颖的 CaM 监管假设： (1) CaM 通过与 KCNQ1 胞质和跨膜同时相互作用来调节 KCNQ1 域，(2) CaM 对不同的 KCNQ1 开放状态进行选择性调节，(3) CaM 调节需要 concurrent ATP binding.目前，几乎没有功能数据支持这些假设作为真正的门控 机制。该提案计划研究这些新颖的 CaM 监管假设，具有三个目标。目标1 将表征破坏 CaM 与跨膜同时相互作用的功能影响， 具有系统诱变、电生理记录和光学测定的细胞质结构域。 Aim 2 将利用生物物理方法来探讨 CaM 通过以下方式改变 KCNQ1 电流的机制假设 通过诱变和蛋白质工程方法选择性调节不同的 KCNQ1 开放状态。目标 3 将扩大 KCNQ1 多配体监管范围。目标3将回答CaM是否对KCNQ1进行调节 需要结合电生理学和结合的 ATP 与 KCNQ1 细胞质结构域同时结合 fluorescence techniques.这项研究的结果将在功能上建立 CaM 调节的新机制 KCNQ1，确定 KCNQ1 和 CaM 中的哪些突变以及如何因 CaM 失调而导致 LQTS。这些 研究结果将为先天性 LQTS 的精准医学方法铺平道路，并可能揭示治疗方法 通过针对这些新颖的 CaM 调节机制来对抗 LQTS。