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; 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，心脏中缓慢激活的延迟整流钾（K+）电流至关重要 生理学是其α（KCNQ1）或β（KCNE1）亚基中突变的证据 与多个心律不齐综合症有关，包括长QT综合征（LQT）；短QT综合征；和 家族性心房颤动。 PKA在同情刺激期间更新IKS频道 磷酸化，对心脏作用电位的物理缩短造成了巨大贡献 对交感神经活动的反应。这种缩短是确保足够的心室填充时间的必要缩短 随着心率的增加。同时刺激也是大多数突然的死亡 从LQT出发。了解这些突变引起的心律不齐综合征的机制 需要在正常和 疾病改变了IKS通道。但是迄今为止，KCNE1如何改变KCNQ1频道门控的关键问题 以及如何通过PKA调制的IKS频道仍未完全回答。先前的研究表明可能 肾上腺响应期间KCNQ1的N末端与C-terminus之间的相互作用。这里， 我们将使用荧光非天然氨基酸作为FRET实验的基础 在有或没有肾上腺素挑战的情况下，这些关键的细胞内结构域中。我们以前的工作已经揭示了 通道的β-AR调节需要组装大分子复合物，包括两个KCNQ1 和KCNE1以及衔接蛋白Yotiao（AKAP 9）。我们将在这里使用新颖的纳米生物来交付 PKA的调节域直接与AKAP9共同组装，直接向KCNQ1/KCNE1通道。 这些实验将使AKAP9在传递信号分子中的关键作用中解剖 kcnq1/kcne1来自AKAP的其他假定调节作用，以调制通道功能 磷酸化。在KCNQ1和KCNQ1和 VSD和PD之间的KCNE1图表明KCNE1位于KCNQ1结构的该区域。 我们将测试使用S6中不同门控铰链打开KCNQ1和KCNQ1/KCNE1通道。我们将 这里还确定KCNQ1-KCNE1相互作用残差，并确定这些残差是否影响 不同的门控铰链。 PKA已显示会改变电压依赖性，亚电导占用率， 和IKS频道的动力学。使用电压夹具荧光法以及突变和PIP2部署 揭开VSD和PD的状态，我们将确定PKA是否影响VSD，PD和/或VSD-TO-PD耦合 IKS频道。这些实验的预期结果将为PKA和PKA控制提供结构性基础 该关键离子通道的物理功能的KCNE1，还将为 开发药物以调节其活性。这将是建立特定于突变治疗的里程碑 由KCNQ1和KCNE1突变引起的疾病，例如心律不齐。