Modulation of KCNQ1 channel activity

KCNQ1 通道活性的调节

• 批准号: 9899256
• 负责人: ROBERT S KASS
• 金额: $ 44.1万
• 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/9899256
• 关键词: A kinase anchoring protein; AKAP9 gene; Action Potentials; Adaptor Signaling Protein; Adenylate Cyclase; Adrenergic Agents; Affect; Area; Arrhythmia; Basic Science; Binding; Biological Assay; Cardiac; Cardiac Myocytes; Clinical; Complex; Coupling; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Dependence; Disease; Dissection; Ensure; Environment; Familial atrial fibrillation; Fluorescence Resonance Energy Transfer; Fluorometry; Future; Heart; Heart Rate; Human; Induced Mutation; Ion Channel; Kinetics; Life; Link; Long QT Syndrome; Macromolecular Complexes; Maps; Measurement; Membrane Potentials; Methodology; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; Nerve; PDE4D3 phosphodiesterase; Pathologic; Pharmacology Study; 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; improved; mutant; nanobodies; novel; response; unnatural amino acids; voltage; voltage clamp

IKS, 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 IKS 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 IKS channels. But to date, the critical questions of how KCNE1 alters KCNQ1 channel gating and how IKS channels are modulated by PKA are still not fully answered. Previous studies suggest a possible interaction between the N-terminus of KCNQ1 and C-terminus of KCNE1 during adrenergic responses. Here, we will use fluorescent unnatural amino acids as the basis for FRET experiments that will assay the proximity of these critical intracellular domains with and without adrenergic challenge. Our previous work has revealed that β-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 IKS 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 IKS 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.

IKS是心脏缓慢激活的延迟整流钾（delayed rectifier potassium，K+）电流，对人的心脏有重要意义 从其α（KCNQ 1）或β（KCNE 1）亚基的突变已经被证实的事实中可以看出， 与多种心律失常综合征相关，包括长QT综合征（LQTS）;短QT综合征;以及 家族性房颤IKS通道在PKA刺激交感神经过程中上调 磷酸化，这对心脏动作电位的生理性缩短起着关键作用， 对交感神经活动的反应。这种缩短是必要的，以确保足够的心室充盈时间 伴随着心率的增加。也正是在交感神经刺激期间， LQTS发生。了解这些突变引起的心律失常综合征的机制 需要解开KCNQ 1和KCNE 1之间的分子相互作用， 疾病改变了IKS通道。但迄今为止，KCNE 1如何改变KCNQ 1通道门控的关键问题 IKS通道是如何被PKA调节的，目前还没有完全的答案。先前的研究表明， 肾上腺素能反应中KCNQ 1的N-末端和KCNE 1的C-末端之间的相互作用。在这里， 我们将使用荧光非天然氨基酸作为FRET实验的基础， 这些关键的细胞内结构域有和没有肾上腺素能的挑战。我们之前的研究显示 β-AR对通道的调节需要一种大分子复合物的组装， 和KCNE 1，以及衔接蛋白Yotiao（AKAP 9）。我们将在这里使用新的纳米抗体， PKA的调节结构域直接连接到KCNQ 1/KCNE 1通道，有或没有与AKAP 9共组装。 这些实验将允许解剖AKAP 9在将信号分子递送到细胞中的关键作用。 KCNQ 1/KCNE 1从AKAP在调节通道功能后的额外假定调节作用 磷酸化在最近的KCNQ 1的CryoEM结构中，KCNQ 1和 KCNE 1位于VSD和PD之间，表明KCNE 1位于KCNQ 1结构的这一区域。 我们将在S6中测试KCNQ 1和KCNQ 1/KCNE 1通道是否使用不同的门控铰链打开。我们将 本文还鉴定了KCNQ 1-KCNE 1相互作用的残基，并确定这些残基是否影响 不同的门铰PKA已被证明可以改变电压依赖性，亚电导占有率， 和IKS通道的动力学。使用电压钳荧光测定法以及突变和PIP 2缺失， 将VSD和PD解偶联，我们将确定PKA是否影响VSD、PD和/或VSD-PD偶联， IKS频道。这些实验的预期结果将为PKA调控提供结构基础， 研究KCNE 1这一重要的离子通道的生理功能，也将为研究KCNE 1的作用机制提供新的靶点。 开发药物以调节其活性。这将是针对突变特异性治疗的里程碑。 由KCNQ 1和KCNE 1突变引起的疾病，如心律失常。