Calmodulin Regulation of KCNQ1 Potassium Channels

项目摘要 在心脏中，KCNQ1电压门控钾通道形成缓慢延迟整流器的α亚单位 钾电流，一种对心脏动作电位终止至关重要的复极化电流。钙调素（CaM）是一种 结合KCNQ1胞质结构域并调节正常KCNQ1的专性KCNQ1辅助亚基 贩运和功能。KCNQ1（>600）和CaM的遗传突变导致先天性长QT综合征 （LQTS），其使患者易患心律失常和心源性猝死。关于这一点，我们知之甚少。 特定的KCNQ1突变或KCNQ1的CaM失调引起的分子机制 LQTS。相反，由于钙调素在心肌细胞中结合多种离子通道靶点， CaM突变通过诱导KCNQ1功能障碍引起LQTS。本研究的总体目标是阐明 CaM调节KCNQ1的机制，并确定KCNQ1和CaM中的致突变突变， 特别是通过钙调素失调引起LQTS。直到最近，KCNQ1结构和门控的缺乏 机制的见解阻碍了CaM调节KCNQ1的机制研究。但最近的 KCNQ1功能和结构研究的突破揭示了新的发现， 这个屏障。该提议的动机是来自这些研究的三个新的CaM调节假设： (1)CaM通过与KCNQ1的胞质和跨膜相互作用调节KCNQ1 结构域，（2）CaM对不同的KCNQ1开放状态进行选择性调节，（3）CaM调节需要 同时ATP结合。目前，几乎没有功能数据支持这些假设为真正的门控 机制等该提案计划调查这些新的钙调素调控假说有三个目标。要求1 将表征破坏钙调素与跨膜蛋白的同时相互作用的功能影响， 用系统诱变、电生理学记录和光学测定来研究细胞质结构域。目的2 将利用生物物理学的方法来探讨CaM改变KCNQ 1电流的机制假说， 用诱变和蛋白质工程方法选择性调节不同的KCNQ1开放状态。目标3 将范围扩大到多配体KCNQ1调节。目标3将回答CaM是否调节KCNQ1 需要同时结合ATP与KCNQ1胞质结构域，并结合电生理学， 荧光技术本研究的结果将在功能上建立新的钙调素调节机制 的KCNQ1，确定KCNQ1和CaM的突变以及如何导致LQTS由于CaM失调。这些 研究结果将为先天性LQTS的精确医学方法铺平道路，并可能揭示治疗方法。 通过靶向这些新的CaM调节机制来对抗LQTS的方法。

PROJECT SUMMARY In the heart, KCNQ1 voltage-gated potassium channels form the alpha-subunits for the slow delayed rectifier potassium current, a repolarizing current critical for cardiac action potential termination. Calmodulin (CaM) is an obligatory KCNQ1 auxiliary subunit which binds the KCNQ1 cytoplasmic domain and regulates normal KCNQ1 trafficking and function. Inherited mutations in KCNQ1 (>600) and CaM lead to congenital long QT syndrome (LQTS), which predisposes patients to arrhythmias and sudden cardiac death. Little is known regarding the specific KCNQ1 mutations or the molecular mechanism underlying CaM dysregulation of KCNQ1 to cause LQTS. Conversely, because CaM binds multiple ion channel targets in cardiomyocytes, it is unclear whether CaM mutations cause LQTS by inducing KCNQ1 dysfunction. This study’s overall goal is to elucidate mechanisms of CaM regulation of KCNQ1 and identify arrhythmogenic mutations in KCNQ1 and CaM which cause LQTS specifically through CaM dysregulation. Until recently, the lack of KCNQ1 structural and gating mechanism insights has hindered mechanistic studies of CaM regulation of KCNQ1. However, recent breakthroughs in both functional and structural studies of KCNQ1 revealed novel findings which may overcome this barrier. This proposal is motivated by three novel CaM regulatory hypotheses derived from these studies: (1) CaM regulates KCNQ1 through simultaneous interactions with the KCNQ1 cytoplasmic and transmembrane domains, (2) CaM exerts selective regulation on distinct KCNQ1 open states, and (3) CaM regulation requires concurrent ATP binding. Presently, there is little functional data to support these hypotheses as genuine gating mechanisms. This proposal plans to investigate these novel CaM regulatory hypotheses with three aims. Aim 1 will characterize the functional impact of disrupting CaM simultaneous interactions with the transmembrane and cytoplasmic domains with systematic mutagenesis, electrophysiological recordings, and optical assays. Aim 2 will utilize a biophysical approach to probe the mechanistic hypothesis that CaM alters KCNQ1 current through selective regulation of distinct KCNQ1 open states with mutagenesis and protein engineering methods. Aim 3 will expand the scope to multi-ligand KCNQ1 regulation. Aim 3 will answer whether CaM regulation of KCNQ1 requires concurrent ATP binding to the KCNQ1 cytoplasmic domain with combined electrophysiology and fluorescence techniques. Results from this study will functionally establish novel mechanisms of CaM regulation of KCNQ1, identify which and how mutations in KCNQ1 and CaM lead to LQTS due to CaM dysregulation. These findings will pave the way for a precision medicine approach to congenital LQTS and may reveal therapeutic approaches against LQTS through targeting these novel CaM regulatory mechanisms.