Real-time potassium channel subunit dynamics

项目摘要 本论文主要研究KCNE家族离子通道参与的大分子信号复合物 调节（β）亚基。KCNE亚基是单次跨膜β亚基，已知用于修饰 组织中电压门控性钾通道α亚基如KCNQ 1的功能特性 包括听觉系统和心肌细胞。五种人类KCNE亚基中的每一种都可以调节 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， 与其他亚基组合物相比，此外，已知的40种Kv α亚基中， 在人类基因组中，已知受一种以上KCNE同种型调节。许多这样的复合体 已经被确定，它们在哺乳动物生理学中的绝对必要性通过功能研究得到阐明， 与人类或小鼠遗传学结合，或在某些情况下与两者结合。尽管它们对关键的 生物学过程和与使人衰弱的疾病的联系，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疗效，基本 问题围绕化学计量，亚基动力学，和组成的灵活性，含KCNE 复杂的问题仍然没有得到解决。此外，我们最近发现，KCNQ 1-KCNE 2钾 通道与几种钠偶联的溶质转运蛋白形成钙离子调节复合物-此外， 一类新的信号复合物，目前对它的了解甚至更少。为了解决这些主要问题， 知识的差距，在拟议的项目中，我们将采用尖端的荧光动力学技术， 使细胞表面的通道复杂动力学可视化，并测试新的和重要的假设 这是由实地调查人员提出的，但没有直接测试。在目标1中，我们将使用TIRF， 图像均方位移（iMSD）和数量和亮度分析，以测试长期存在的 假设KCNQ 1通道可以在细胞表面丢失或获得KCNE亚基，并阐明了 各种含KCNE的钾通道复合物的亚基化学计量，包括形成的那些 与溶质转运蛋白。在目标2中，我们将使用TIRF，共聚焦显微镜，互相关光栅扫描图像， 相关光谱（ccRICS）和iMSD来阐明KCNQ 1复合物是否可以包含超过 一次一个KCNE亚型，以及这些新的KCNE亚基是否可以加入现有的KCNE亚基， 在细胞表面与KCNQ 1复合。利用和开发新的方法来应对 关于基于KCNE的渠道的动态能力的长期问题将提供前所未有的 关于这类广泛存在的离子通道的信息对听觉，心脏， 和其他组织。此外，优化这些方法来解决Kv-KCNE复合物也将开放 这些技术，以回答类似的问题，其他离子通道和多亚基膜蛋白， 将军

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.