Molecular Physiology of KCNE Potassium Channel Subunits

KCNE 钾通道亚基的分子生理学

• 批准号: 6923262
• 负责人: Alfred L. George
• 金额: $ 36.51万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923262
• 关键词: RNA interference; SDS polyacrylamide gel electrophoresis; cardiac myocytes; cardiovascular disorder risk; clinical research; dogs; electrophysiology; gene expression; gene induction /repression; gene interaction; heart disorder; heart function; human tissue; immunocytochemistry; immunoprecipitation; laboratory mouse; membrane proteins; molecular pathology; pore forming protein; potassium channel; regulatory gene; tissue /cell culture

DESCRIPTION (provided by applicant): Voltage-gated potassium (Kv) channels are modulated by at least three distinct classes of accessory proteins including the KCNE family of single transmembrane proteins. In the human genome, KCNE proteins are encoded by five genes designated KCNE1 through KCNE5. The physiological importance of three KCNE genes has been demonstrated by associations with inherited disorders of cardiac and skeletal muscle excitability. Mutation of KCNE 1 impairs generation of the slowly activating cardiac delayed rectifier current (IKS), a critical potassium current contributing to myocardial repolarization that can be reconstituted in vitro by heterologous co-expression of recombinant KCNQ1 potassium channels with KCNE1. Other KCNE proteins also exert diverse functional effects on heterologously expressed KCNQ1. Co-expression of KCNQ1 with KCNE3, KCNE4 or KCNE5 is particularly interesting with functional effects ranging from constitutive activation (KCNE3) to suppression (KCNE4, KCNE5). We have recently demonstrated that all KCNE genes are expressed in human cardiac myocytes to varying degrees. We have further demonstrated that KCNE4 and KCNE5 exert potent inhibitory influences on heterologously expressed IKappaS and KCNE4 appears dominant over KCNE1 in this context. In addition to potential interactions with KCNQ1, KCNE proteins affect the functional properties of other cloned potassium channels. The physiological relevance of these in vitro observations remains uncertain. The observations that multiple KCNE proteins can modulate KCNQ1 channels to produce diverse biophysical phenotypes raises several intriguing questions with important implications for understanding the regulation of IKS as well as other potassium currents in normal and diseased heart. Can different KCNE subtypes compete functionally with KCNE1 for modulation of KCNQ1? Can KCNQ1 associate with more than one KCNE subtype in the same cell and at the same time? Do KCNE4 and KCNE5 participate in the pathophysiology of heart failure and other arrhythmia-prone conditions? In this proposal, we outline several experimental approaches for defining the physiological importance of KCNE subunits with a particular focus on KCNE4 and KCNE5. In Specific Aim 1, we will use a heterologous expression system to assess the range of functional and biochemical interactions possible between KCNQ1 and different KCNE subunits with a focus on KCNE4 and KCNE5. In Specific Aim 2, we will test the hypothesis that KCNE4 and KCNE5 are physiologically significant regulators of potassium currents in native cardiac myocytes by using two independent gene silencing approaches. Finally in Specific Aim 3, we will examine expression patterns of KCNQ1 and KCNE genes in diseased human heart and in an experimental canine model of ventricular arrhythmia susceptibility. Results from these studies will have implications for advancing our knowledge of the complex interplay between pore-forming and accessory subunits that comprise human potassium channels expressed in the heart and in other tissues.

描述（由申请人提供）：电压门控钾（Kv）通道由至少三种不同类别的辅助蛋白调节，包括单跨膜蛋白的 KCNE 家族。在人类基因组中，KCNE 蛋白由 KCNE1 至 KCNE5 五个基因编码。三个 KCNE 基因的生理重要性已通过与心脏和骨骼肌兴奋性遗传性疾病的关联得到证实。 KCNE 1 的突变会损害缓慢激活的心脏延迟整流电流 (IKS) 的产生，这是一种有助于心肌复极的关键钾电流，可以通过重组 KCNQ1 钾通道与 KCNE1 的异源共表达在体外重建。其他 KCNE 蛋白也对异源表达的 KCNQ1 发挥不同的功能作用。 KCNQ1 与 KCNE3、KCNE4 或 KCNE5 的共表达特别令人感兴趣，其功能作用范围从组成型激活 (KCNE3) 到抑制 (KCNE4、KCNE5)。我们最近证明，所有 KCNE 基因在人类心肌细胞中都有不同程度的表达。我们进一步证明 KCNE4 和 KCNE5 对异源表达的 IKappaS 发挥有效的抑制作用，并且在这种情况下 KCNE4 似乎比 KCNE1 更占优势。除了与 KCNQ1 的潜在相互作用外，KCNE 蛋白还影响其他克隆钾通道的功能特性。这些体外观察结果的生理相关性仍不确定。 多个 KCNE 蛋白可以调节 KCNQ1 通道以产生不同的生物物理表型的观察结果提出了几个有趣的问题，对于理解正常和患病心脏中 IKS 以及其他钾电流的调节具有重要意义。不同的 KCNE 亚型能否在功能上与 KCNE1 竞争 KCNQ1 的调节？ KCNQ1 能否同时与同一细胞中的多个 KCNE 亚型相关？ KCNE4 和 KCNE5 是否参与心力衰竭和其他心律失常易发病症的病理生理学？ 在本提案中，我们概述了几种定义 KCNE 亚基生理重要性的实验方法，特别关注 KCNE4 和 KCNE5。在具体目标 1 中，我们将使用异源表达系统来评估 KCNQ1 和不同 KCNE 亚基之间可能的功能和生化相互作用的范围，重点是 KCNE4 和 KCNE5。在具体目标 2 中，我们将通过使用两种独立的基因沉默方法来测试 KCNE4 和 KCNE5 是天然心肌细胞中钾电流的生理学重要调节剂的假设。最后，在具体目标 3 中，我们将检查患病人类心脏和室性心律失常易感性实验犬模型中 KCNQ1 和 KCNE 基因的表达模式。这些研究的结果将有助于加深我们对成孔亚基和辅助亚基之间复杂相互作用的认识，这些亚基构成了在心脏和其他组织中表达的人类钾通道。