Investigation of the Role of the KCNH Voltage-Gated Potassium Channel Intracellular Domains in Gating

KCNH 电压门控钾通道细胞内结构域在门控中的作用研究

• 批准号: 10393985
• 负责人: Whitney Alexandra Stevens-Sostre
• 金额: $ 3.55万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393985
• 关键词: Action Potentials; Address; Animal Model; Area; Arrhythmia; Award; Binding; Binding Sites; Biophysics; Brain; C-terminal; Calcium; Calmodulin; Cardiac; Cardiac Death; Collaborations; Complex; Couples; Critical Thinking; Cryoelectron Microscopy; Cyclic Nucleotides; Development; Disease; Doctor of Philosophy; Electrodes; Electron Microscope; Electrophysiology (science); Epilepsy; Ethers; Family; Functional disorder; Future; Future Generations; Gap Junctions; Genes; Goals; Heart; Human; Hydrophobic Interactions; Hydrophobicity; Image; Investigation; Ion Channel; Ion Channel Protein; Journals; Kinetics; Knowledge; Laboratories; Laboratory Research; Learning; Life; Light; Malignant Neoplasms; Maps; Measures; Mediating; Memory; Methods; Molecular; Molecular Machines; Mutation; N-terminal; Nature; Nervous system structure; Neurologic; Neurological Models; Neurons; Neurosciences; Nucleotides; Oral; Oxidation-Reduction; Oxygen; Pathologic Processes; Pathway Analysis; Pathway interactions; Peer Group; Pharmaceutical Preparations; Phase; Physiological; Physiological Processes; Point Mutation; Positioning Attribute; Potassium Channel; Presynaptic Terminals; Process; Property; Publications; Rattus; Research; Research Personnel; Research Project Grants; Role; Schizophrenia; Scientist; Shapes; Site-Directed Mutagenesis; Structure; Synaptic Transmission; Techniques; Testing; Thermodynamics; Training; Ventricular; Voltage-Gated Potassium Channel; Work; Xenopus oocyte; alpha helix; biophysical techniques; diagnostic biomarker; experience; insight; meetings; mutant; nervous system disorder; neuronal excitability; neurotransmitter release; novel; optogenetics; posters; protein protein interaction; sensor; simulation; skills; synaptic function; targeted treatment; therapeutic development; voltage; voltage clamp

PROJECT SUMMARY/ABSTRACT KCNH channels, also known as the Ether-à-go-go (Eag) family, are voltage-gated potassium ion channels that have roles in neuronal excitability and cardiac repolarization. The dysfunction of these ion channels is implicated in a variety of diseases, including cancer, epilepsy, and cardiac arrythmia, making them promising targets for both diagnostic markers and the development of therapeutic drugs. KCNH channels like EAG and human Ether-à-go-go–Related Gene (hERG) possess unique and highly conserved intracellular domains that have evolved to serve unique physiological roles. The N-terminal Per-Arnt-Sim (PAS) domain has diverse functions in nature: they serve as input modules that mediate protein-protein interactions and as redox potential, oxygen, and light sensors. The C-terminal cyclic nucleotide binding homology (CNBh) domain has structural homology to cyclic nucleotide binding (CNB) domains but lack the ability to bind nucleotides. The intracellular PAS and CNBh domains modulate gating in KCNH channels and are known from structural and functional studies to associate in a complex. Several disease-associated mutations are concentrated at the PAS-CNBhD interface, highlighting the physiological importance of their interaction. However, details of how these domains interact to allosterically regulate critical channel functions, such as slow deactivation in hERG and calmodulin (CaM) inhibition in EAG1, are not understood. This proposal seeks to investigate the role of the KCNH channel intracellular domains in gating by combining structural, computational, and electrophysiological approaches. In Aim 1 (PhD Progress), I describe the presence of a hydrophobic interaction among residues in the PAS-cap (residues1-25), the downstream globular PAS (residues 26-135), and the CNBh domain of a neighboring subunit that are critical for slow deactivation in hERG channels. In Aim 2 (F99 Phase), I propose to uncover the allosteric pathways that mediate Ca2+-CaM inhibition of EAG1 currents. Given the importance of KCNH channels in various physiological and pathological processes, we expect our novel findings to have broad implications in neuroscience and beyond. During the F99 Phase, I will continue my thesis work in my advisor's state-of-the art laboratory with the guidance and support of my colleagues and graduate trainee peer group. I will learn cutting-edge techniques and methods of analysis, as well as develop my critical thinking skills to interpret my findings. I will continue engaging in professional development activities, including journal clubs, seminars, and lab meetings. Finally, I will share my scientific findings through publications, and oral and poster presentations at scientific meetings. My long-term goal is to understand the effects of channelopathies within the nervous system. Thus, for the K00 phase, I have identified specific areas, concepts, and techniques I must develop to become a well-rounded researcher and expert in neurological channelopathies. I intend to utilize my expertise to establish a diverse and equitable academic research laboratory to train future generations of scientists.

项目概要/摘要 KCNH 通道，也称为 Ether-à-go-go (Eag) 系列，是电压门控钾离子通道， 在神经元兴奋性和心脏复极中发挥作用。这些离子通道的功能障碍是 与多种疾病有关，包括癌症、癫痫和心律失常，使其具有广阔的前景 诊断标记物和治疗药物开发的目标。 KCNH 频道，例如 EAG 和 人类 Ether-à-go-go 相关基因 (hERG) 拥有独特且高度保守的细胞内结构域， 已经进化出独特的生理作用。 N 端 Per-Arnt-Sim (PAS) 域具有多种 自然界中的功能：它们作为介导蛋白质-蛋白质相互作用的输入模块和氧化还原作用 电位、氧气和光传感器。 C 端环核苷酸结合同源 (CNBh) 结构域具有 与环核苷酸结合（CNB）结构域具有结构同源性，但缺乏结合核苷酸的能力。这 细胞内 PAS 和 CNBh 结构域调节 KCNH 通道中的门控，并且从结构和 关联于复合体的功能研究。一些与疾病相关的突变集中在 PAS-CNBhD 界面，强调了它们相互作用的生理重要性。不过具体如何操作的细节 这些结构域相互作用以变构调节关键通道功能，例如 hERG 中的缓慢失活 EAG1 中的钙调蛋白 (CaM) 抑制作用尚不清楚。该提案旨在调查 通过结合结构、计算和电生理学来门控 KCNH 通道细胞内结构域 接近。在目标 1（博士进展）中，我描述了残基之间存在疏水相互作用。 PAS-cap（残基 1-25）、下游球状 PAS（残基 26-135）和 CNBh 结构域 相邻亚基对于 hERG 通道缓慢失活至关重要。在目标 2（F99 阶段）中，我建议 揭示介导 Ca2+-CaM 对 EAG1 电流抑制的变构途径。鉴于重要性 KCNH 在各种生理和病理过程中发挥作用，我们希望我们的新发现能够 在神经科学及其他领域具有广泛的影响。 在 F99 阶段，我将在我导师最先进的实验室中继续我的论文工作 我的同事和研究生实习生同行群体的指导和支持。我会学习最前沿的技术 和分析方法，以及培养我的批判性思维能力来解释我的发现。我会继续 参与专业发展活动，包括期刊俱乐部、研讨会和实验室会议。最后，我 将通过出版物以及科学会议上的口头和海报展示分享我的科学发现。 我的长期目标是了解神经系统内通道病的影响。因此，对于 K00 阶段，我已经确定了我必须发展的特定领域、概念和技术，以成为一名全面发展的人 神经通道病的研究员和专家。我打算利用我的专业知识建立一个多元化的 和公平的学术研究实验室来培养下一代科学家。