Modulation of KCNQ1 channel activity

KCNQ1 通道活性的调节

• 批准号: 10079488
• 负责人: ROBERT S KASS
• 金额: $ 40.83万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10079488
• 关键词: A kinase anchoring protein; AKAP9 gene; Action Potentials; Adaptor Signaling Protein; Adenylate Cyclase; Affect; Area; Arrhythmia; Basic Science; Binding; Cardiac; Cardiac Myocytes; Clinical; Complex; Coupling; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Dependence; Disease; Dissection; Ensure; Familial atrial fibrillation; Fluorometry; Heart; Heart Rate; Human; Induced Mutation; Ion Channel; Kinetics; Life; Link; Long QT Syndrome; Macromolecular Complexes; Maps; Membrane Potentials; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; Nerve; PDE4D3 phosphodiesterase; Pathologic; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Physiological; Physiology; Potassium; Potassium Channel; Protein phosphatase; Proteins; Regulation; Role; Short QT syndrome; Signal Transduction; Signaling Molecule; Structure; Sudden Death; Syndrome; Testing; Time; Translating; Variant; Ventricular; Work; beta-adrenergic receptor; drug development; experimental study; mutant; nanobodies; novel; public health relevance; response; voltage; voltage clamp

Modified Project Summary/Abstract Section I{KS}, the slowly activating delayed rectifier potassium (K[+]) current in the heart is critical importance to human physiology as evident from the fact that mutations in either its α (KCNQ1) or β (KCNE1) subunit have been linked to multiple cardiac arrhythmia syndromes, including long QT syndrome (LQTS); short QT syndrome; and familial atrial fibrillation. The I{KS} channel is upregulated during sympathetic stimulation by PKA phosphorylation, which contributes critically to the physiological shortening of cardiac action potentials in response to sympathetic nerve activity. This shortening is necessary to ensure adequate ventricular filling time with accompanying increases in heart rate. It is also during sympathetic stimulation that most sudden deaths from LQTS occur. Understanding the mechanisms that underlie these mutation-induced arrhythmia syndromes requires unraveling the molecular interactions between KCNQ1 and KCNE1 within the context of normal and disease altered I{KS} channels. But to date, the critical questions of how KCNE1 alters KCNQ1 channel gating and how I{KS} channels are modulated by PKA are still not fully answered. Our previous work has revealed that Beta-AR regulation of channels requires assembly of a macromolecular complex that includes both KCNQ1 and KCNE1, as well as the adaptor protein Yotiao (AKAP 9). We will here use novel nanobodies to deliver regulatory domains of PKA directly to the KCNQ1/KCNE1 channel with and without co-assembly with AKAP9. These experiments will allow dissection of the critical role of AKAP9 in the delivery of signaling molecules to KCNQ1/KCNE1 from additional putative modulatory roles of the AKAP in modulating channel function post phosphorylation. In the recent CryoEM structure of KCNQ1 putative interacting residues between KCNQ1 and KCNE1 map between the VSD and PD, suggesting that KCNE1 is located in this area of the KCNQ1 structure. We will test whether KCNQ1 and KCNQ1/KCNE1 channels open using different gating hinges in S6. We will here also identify KCNQ1-KCNE1 interacting residues and determine whether these residues affect the different gating hinges. PKA has been shown to alter the voltage dependence, sub-conductance occupancy, and kinetics of I{KS} channels. Using voltage clamp fluorometry together with mutations and PIP2 depletion that uncouple the VSD and PD, we will determine whether PKA affect the VSD, PD, and/or VSD-to-PD coupling in I{KS} channels. The anticipated results of these experiments will provide a structural basis for control by PKA and KCNE1 of the physiological function of this critical ion channel and will also provide novel targets for the development of drugs to modulate its activity. This would be a milestone toward mutation-specific treatments of diseases, such as cardiac arrhythmias, caused by mutations in KCNQ1 and KCNE1.

修改后的项目摘要/摘要部分 I{KS}，心脏中缓慢激活的延迟整流钾 (K[+]) 电流对人类生理学至关重要，这一点可以从其 α (KCNQ1) 或 β (KCNE1) 亚基的突变与其相关的事实中看出多种心律失常综合征，包括长 QT 综合征 (LQTS) 和家族性短 QT 综合征；PKA 交感神经刺激期间 I{KS} 通道上调。磷酸化对交感神经活动引起的心脏动作电位的生理缩短至关重要，这种缩短对于确保足够的心室充盈时间以及伴随的心率增加是必要的。大多数 LQTS 猝死也是在交感神经刺激期间发生的。了解这些突变诱发的心律失常综合征的机制需要揭示正常和疾病改变的 I{KS} 通道中 KCNQ1 和 KCNE1 之间的分子相互作用。 KCNE1 如何改变 KCNQ1 通道门控以及 I{KS} 通道如何被 PKA 调制的问题仍然没有得到完全解答。 Beta-AR 通道调节需要组装包含 KCNQ1 和 KCNE1 以及接头蛋白 Yotiao (AKAP 9) 的大分子复合物，我们将在此使用新型纳米抗体将 PKA 的调节域直接传递至 KCNQ1/KCNE1 通道。无论是否与 AKAP9 共组装，这些实验将能够剖析 AKAP9 在向 KCNQ1/KCNE1 传递信号分子中的关键作用。来自 AKAP 在磷酸化后调节通道功能中的额外假定调节作用在 KCNQ1 的最新 CryoEM 结构中，KCNQ1 和 KCNE1 之间假定的相互作用残基位于 VSD 和 PD 之间，表明 KCNE1 位于 KCNQ1 结构的该区域。我们将测试 KCNQ1 和 KCNQ1/KCNE1 通道是否使用 S6 中的不同门控铰链打开。 KCNQ1-KCNE1 相互作用残基并确定这些残基是否影响不同的门控铰链，使用电压钳荧光测定法以及突变和 PIP2 耗竭已证明 PKA 会改变 I{KS} 通道的电压依赖性、亚电导占有率和动力学。解耦 VSD 和 PD，我们将确定 PKA 是否影响 I{KS} 通道中的 VSD、PD 和/或 VSD 与 PD 耦合。这些实验将为 PKA 和 KCNE1 控制这一关键离子通道的生理功能提供结构基础，也将为开发调节其活性的药物提供新的靶点，这将是突变特异性治疗的一个里程碑。由 KCNQ1 和 KCNE1 突变引起的疾病，例如心律失常。