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，心脏中缓慢激活的延迟整流钾 (K+) 电流对人类至关重要 其 α (KCNQ1) 或 β (KCNE1) 亚基的突变已被证明是生理学的证据 与多种心律失常综合征有关，包括长 QT 综合征 (LQTS)；以及 家族性心房颤动在 PKA 交感神经刺激过程中 IKS 通道上调。 磷酸化，这对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是必要的。 伴随着心率的增加，大多数猝死也是在交感神经刺激期间发生的。 了解 LQTS 发生的机制。 需要在正常和正常情况下揭示 KCNQ1 和 KCNE1 之间的分子相互作用 但迄今为止，关键问题是 KCNE1 如何改变 KCNQ1 通道门控。 IKS 通道如何被 PKA 调节仍然没有完全解答，之前的研究表明了一种可能。 肾上腺素能反应期间 KCNQ1 的 N 末端和 KCNE1 的 C 末端之间的相互作用。 我们将使用荧光非天然氨基酸作为 FRET 实验的基础，以测定邻近性 我们之前的工作已经揭示了这些关键细胞内结构域在有或没有肾上腺素能挑战的情况下的变化。 通道的 β-AR 调节需要组装包含 KCNQ1 的大分子复合物 和 KCNE1，以及接头蛋白 Yotiao (AKAP 9)，我们将在这里使用新型纳米抗体来递送。 PKA 的调控域直接连接到 KCNQ1/KCNE1 通道，无论是否与 AKAP9 共组装。 这些实验将有助于剖析 AKAP9 在将信号分子传递到细胞中的关键作用。 KCNQ1/KCNE1 来自 AKAP 在调节通道功能后的额外假定调节作用 在最近的 KCNQ1 的 CryoEM 结构中，KCNQ1 和 KCNQ1 之间推定的相互作用残基。 KCNE1图位于VSD和PD之间，表明KCNE1位于KCNQ1结构的这个区域。 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。 此处还鉴定了 KCNQ1-KCNE1 相互作用残基并确定这些残基是否影响 不同的门控铰链已被证明可以改变电压依赖性、亚电导占有率、 使用电压钳荧光测定法以及突变和 PIP2 耗竭的 IKS 通道动力学。 解耦 VSD 和 PD，我们将确定 PKA 是否影响 VSD、PD 和/或 VSD-to-PD 耦合 这些实验的预期结果将为 PKA 和 IKS 通道的控制提供结构基础。 KCNE1 的这一关键离子通道的生理功能，也将为 开发调节其活性的药物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。