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 心脏中缓慢激活的延迟整流钾电流(K[+])对人体生理起着至关重要的作用，其α(KCNQ1)或β(KCNE1)亚基的突变与多种心律失常综合征有关，包括长QT综合征、短QT综合征和家族性心房颤动。在交感神经刺激过程中，PKA的磷酸化上调了I{KS}通道，这在交感神经活动引起的心脏动作电位生理性缩短中起着重要作用。这种缩短是必要的，以确保有足够的脑室充盈时间，并伴随心率的增加。也正是在交感神经刺激期间，LQTS导致的猝死最多。要了解这些突变引起的心律失常综合征的机制，需要在正常和疾病改变的I{KS}通道的背景下解开KCNQ1和KCNE1之间的分子相互作用。但到目前为止，KCNE1如何改变KCNQ1通道门控以及PKA如何调制I{KS}通道的关键问题仍然没有完全得到回答。我们之前的工作揭示了 β-AR对通道的调节需要组装一个包括KCNQ1和KCNE1的大分子复合体，以及接头蛋白Ytiao(AKAP 9)。在这里，我们将使用新的纳米体将PKA的调节结构域直接传递到KCNQ1/KCNE1通道，并与AKAP9共同组装和不组装。这些实验将从AKAP在磷酸化后调节通道功能的额外假定调制作用中剖析AKAP9在向KCNQ1/KCNE1传递信号分子中的关键作用。在最近的KCNQ1的低温EM结构中，推测KCNQ1和KCNE1之间的相互作用残基在VSD和PD之间映射，表明KCNE1位于KCNQ1结构的这一区域。我们将在S6中测试是否使用不同的门铰打开KCNQ1和KCNQ1/KCNE1通道。在这里，我们还将鉴定KCNQ1-KCNE1相互作用的残基，并确定这些残基是否影响不同的门控铰链。PKA已被证明改变I{KS}通道的电压依赖性、亚电导占有率和动力学。利用电压钳荧光法结合突变和PIP2缺失来解偶联VSD和PD，我们将确定PKA是否影响I{KS}通道中的VSD、PD和/或VSD到PD的偶联。这些实验的预期结果将为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。这将是一个里程碑，朝着针对KCNQ1和KCNE1突变引起的心律失常等疾病的突变特异性治疗迈进。