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

项目摘要/摘要 KCNH通道，也称为以太-去-去(EAG)家族，是电压门控钾离子通道， 在神经元兴奋性和心脏复极中起作用。这些离子通道的功能障碍是 与多种疾病有关，包括癌症、癫痫和心脏性心律失常，这使它们很有希望 作为诊断标记物和治疗药物开发的目标。EAG和KCNH这样的频道 人类Ether-à-Go-Go相关基因(HERG)具有独特且高度保守的胞内区 进化出了独特的生理作用。N端Per-Arnt-Sim(PAS)结构域具有多样性 自然界中的功能：它们作为输入模块，调节蛋白质之间的相互作用，并作为氧化还原 电势、氧气和光传感器。C末端环核苷酸结合同源(CNBh)结构域具有 与环核苷酸结合(CNB)结构域结构同源，但缺乏与核苷酸结合的能力。这个 细胞内Pas和CNBh结构域调节KCNH通道的门控，从结构和 将功能研究联系在一个复合体中。几个与疾病相关的突变集中在 PAS-CNBhD接口，强调了它们相互作用的生理重要性。然而，详细说明如何 这些结构域相互作用，以变构方式调节关键的通道功能，如HERG中的缓慢失活 以及钙调蛋白(CaM)在EAG1中的抑制作用，目前还不清楚。这项提案旨在调查 KCNH通过结合结构、计算和电生理在门控中的细胞内结构域 接近了。在目标1(PHD进展)中，我描述了残基之间存在疏水相互作用 Pas-帽(残基1-25)、下游球状Pas(残基26-135)和A 在HERG通道中对缓慢失活起关键作用的相邻亚基。在目标2(F99阶段)中，我建议 揭示介导钙-钙调素抑制EAG1电流的变构通路。鉴于……的重要性 KCNH通道在不同的生理和病理过程中，我们希望我们的新发现有 在神经科学和其他领域的广泛影响。 在F99阶段，我将在我的导师最先进的实验室继续我的论文工作 指导和支持我的同事和毕业实习生同龄人。我要学习尖端技术 和分析方法，以及发展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，包括杂志俱乐部、研讨会和实验室会议。最后，我 将通过出版物以及在科学会议上的口头和海报演示来分享我的科学发现。 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，才能成为一个全面的 神经经络病的研究人员和专家。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，培养未来一代的科学家。

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.