Real-time potassium channel subunit dynamics

实时钾通道亚基动态

• 批准号: 9264256
• 负责人: Geoffrey W Abbott
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-20 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9264256
• 关键词: Address; Auditory; Auditory system; Biological; Biological Process; Biology; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell membrane; Cell physiology; Cell surface; Cells; Characteristics; Charge; Complex; Confocal Microscopy; Coupled; Data; Dependence; Drug Targeting; Elements; Environment; Epithelium; Exhibits; Family; Fluorescence; Fluorescence Microscopy; Gastrointestinal tract structure; Genetic; Heart; Homeostasis; Human; Human Genome; Image; Imagery; In Vitro; Individual; Ion Channel; Ions; Kinetics; Knowledge; Laboratories; Liquid substance; Membrane Proteins; Movement; Mus; Mutation; Nature; Neurons; One-Step dentin bonding system; Outcome; Pharmacology; Physiology; Potassium; Potassium Channel; Process; Property; Protein Isoforms; Proteins; Regulation; Reporting; Research Personnel; Role; Scanning; Series; Signal Transduction; Sodium; Specificity; Spectrum Analysis; Techniques; Testing; Time; Tissues; base; deafness; experience; flexibility; heart rhythm; human disease; human tissue; in vivo; novel; novel strategies; solute; spectroscopic imaging; stoichiometry; trafficking; voltage

Project Summary This proposal is centered upon macromolecular signaling complexes involving KCNE family ion channel regulatory (β) subunits. The KCNE subunits are single-pass transmembrane β subunits known for modifying the functional properties of voltage-gated potassium (Kv) channel α subunits such as KCNQ1, in tissues including the auditory system and cardiac myocytes. Each of the five human KCNE subunits can regulate multiple different Kv channel α subunits, typically forming heteromeric complexes with unique functional attributes compared to those of other subunit compositions. In addition, many of the forty known Kv α subunits in the human genome are known to be regulated by more than one KCNE isoform. Numerous such complexes have been identified and their absolute necessity in mammalian physiology elucidated by functional studies in combination with either human or mouse genetics, or in some cases both. Despite their necessity for crucial biological processes and linkage to debilitating human diseases, and the potential to leverage KCNE subunit influence on pharmacology to increase the specificity and efficacy of channel-targeted drugs, fundamental questions surrounding the stoichiometry, subunit dynamics, and compositional flexibility of KCNE-containing complexes remain unanswered. In addition, we recently discovered that the KCNQ1-KCNE2 potassium channel forms reciprocally regulating complexes with several sodium-coupled solute transporters – a further, novel class of signaling complexes about which even less is currently understood. To address these major gaps in knowledge, in the proposed project we will employ cutting-edge fluorescence dynamics techniques to enable visualization of channel complex dynamics at the cell surface, and test novel and important hypotheses that have been suggested by investigators in the field, but not directly tested. In Aim 1 we will employ TIRF, image Mean Square Displacement (iMSD) and Number and Brightness analysis to test the longstanding hypothesis that KCNQ1 channels can lose or gain KCNE subunits at the cell surface, and also elucidate subunit stoichiometry for a variety of KCNE-containing potassium channel complexes, including those formed with solute transporters. In Aim 2, we will use TIRF, confocal microscopy, cross-correlation raster-scan image correlation spectroscopy (ccRICS) and iMSD to elucidate whether KCNQ1 complexes can contain more than one KCNE isoform at a time, and whether these new KCNE subunits can join existing KCNE subunits in complexes with KCNQ1 at the cell surface. Harnessing and developing new approaches to answer longstanding questions about the dynamic capabilities of KCNE-based channels will deliver unprecedented information about this widespread class of ion channels crucial to the healthy functioning of auditory, cardiac, and other tissues. In addition, optimization of these approaches to tackle Kv-KCNE complexes will also open up these techniques to answer similar questions for other ion channels and multi-subunit membrane proteins in general.

项目概要 该提案以涉及 KCNE 家族离子通道的大分子信号复合物为中心 KCNE 亚基是单程跨膜 β 亚基，以修饰作用而闻名。 组织中电压门控钾 (Kv) 通道 α 亚基（例如 KCNQ1）的功能特性 包括听觉系统和心肌细胞的五个人类 KCNE 亚基中的每一个都可以调节。 多个不同的 Kv 通道 α 亚基，通​​常形成具有独特功能的异聚体复合物 此外，四十种已知的 Kv α 亚基中的许多都具有与其他亚基组成相比的属性。 已知人类基因组中的 KCNE 亚型受多种此类复合物的调节。 已被鉴定，并且通过功能研究阐明了它们在哺乳动物生理学中的绝对必要性 与人类或小鼠遗传学的结合，或在某些情况下两者的结合，尽管它们至关重要。 生物过程和与人类衰弱疾病的联系，以及利用 KCNE 亚基的潜力 对药理学的影响，以增加通道靶向药物的特异性和功效，根本性 围绕含 KCNE 的化学计量、亚基动力学和组成灵活性的问题 此外，我们最近发现 KCNQ1-KCNE2 钾。 通道与几个钠偶联溶质转运蛋白形成相互调节复合物——进一步， 目前对这些主要信号复合物的了解更少。 由于知识上的差距，在拟议的项目中，我们将采用尖端的荧光动力学技术来 实现细胞表面通道复杂动态的可视化，并测试新颖且重要的假设 已由该领域的研究人员建议，但未直接测试 在目标 1 中，我们将采用 TIRF， 图像均方位移 (iMSD) 以及数量和亮度分析来测试长期存在的 假设 KCNQ1 通道可以在细胞表面失去或获得 KCNE 亚基，并阐明 各种含 KCNE 的钾通道复合物（包括形成的复合物）的亚基化学计量 在目标 2 中，我们将使用 TIRF、共焦显微镜、互相关光栅扫描图像。 相关光谱 (ccRICS) 和 iMSD 阐明 KCNQ1 复合物是否可以包含超过 一次一个 KCNE 同工型，以及这些新的 KCNE 亚基是否可以加入现有的 KCNE 亚基 利用和开发新方法来解决这个问题。 关于基于 KCNE 的渠道的动态能力的长期疑问将带来前所未有的结果 有关这一类广泛存在的离子通道的信息对于听觉、心脏、 此外，针对 Kv-KCNE 复合物的这些方法的优化也将开放。 利用这些技术来回答其他离子通道和多亚基膜蛋白的类似问题 一般的。