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

项目概要/摘要 KCNH通道，也称为Ether-à-go-go（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、癫痫和心律失常，使其具有前景 作为诊断标志物和治疗药物开发的靶点。KCNH频道，如EAG和 人Ether-à-go-go-Related基因（hERG）具有独特且高度保守胞内结构域， 已经进化出了独特的生理功能。N-末端Per-Arnt-Sim（PAS）结构域具有不同的结构域。 在自然界中的功能：它们作为输入模块，介导蛋白质-蛋白质相互作用，并作为氧化还原 电位、氧气和光传感器。C-末端环核苷酸结合同源性（CNBh）结构域具有 与环核苷酸结合（CNB）结构域结构同源，但缺乏结合核苷酸的能力。的 细胞内PAS和CNBh结构域调节KCNH通道门控， 功能研究，以关联在一个复杂的。几种疾病相关的突变集中在 PAS-CNBhD接口，突出了它们相互作用的生理重要性。然而，细节如何 这些结构域相互作用，变构调节关键通道功能，如hERG中的缓慢失活 和钙调蛋白（CaM）抑制EAG 1，不明白。这项建议旨在调查 结合结构、计算和电生理学的门控KCNH通道胞内结构域 接近。在目标1（PhD Progress）中，我描述了在蛋白质中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CNBh结构域， 邻近亚基对于hERG通道中的缓慢失活至关重要。在目的2（F99期），我建议 揭示介导EAG 1电流的Ca 2 +-CaM抑制的变构途径。鉴于必须 KCNH通道在各种生理和病理过程中，我们希望我们的新发现， 在神经科学和其他领域的广泛影响。 在F99阶段，我将继续我的论文工作在我的导师的国家的最先进的实验室与 我的同事和研究生学员同伴团体的指导和支持。我会学习尖端技术 和分析方法，以及发展我的批判性思维能力，以解释我的发现。我将继续 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 我将通过出版物和在科学会议上的口头和海报展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K 00 在这个阶段，我已经确定了我必须发展的特定领域，概念和技术，以成为一个全面的 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样化的 和公平的学术研究实验室，以培养未来的科学家。

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.