MOLECULAR BASIS FOR Kv CHANNEL HETEROGENEITY IN THE HEART

心脏 Kv 通道异质性的分子基础

• 批准号: 7651761
• 负责人: Gea-Ny Tseng
• 金额: $ 47.08万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7651761
• 关键词: 3-Dimensional; Abbreviations; Action Potentials; Affect; Affinity; Age; Aging; Aging-Related Process; Animals; Antibodies; Arrhythmia; Atrial Fibrillation; Binding; Cardiac; Cardiac Myocytes; Cell surface; Chronic; Complementary DNA; Complex; Confocal Microscopy; Data; Electrophysiology (science); Extracellular Domain; Family; Genes; Goals; Hand; Heart; Heart Hypertrophy; Heterogeneity; Human; Hypertension; Immunoblotting; Immunofluorescence Immunologic; Immunoprecipitation; Injection of therapeutic agent; Kinetics; Knowledge; Link; Long QT Syndrome; Modeling; Molecular; Molecular Biology; Molecular Models; Mutation; Nature; Phenotype; Physiologic pulse; Physiological; Play; Potassium; Protein Biochemistry; Proteins; Research Project Grants; Risk; Role; Site; Specific qualifier value; Stress; Structure; Syndrome; System; Therapeutic; Transcript; Transfection; Transmembrane Domain; base; combat; density; design; glycosylation; insight; interdisciplinary approach; interest; member; molecular modeling; novel; pathological aging; public health relevance; research study; response; stoichiometry; trafficking; voltage

DESCRIPTION (provided by applicant): The long-term objectives of this research project are to provide information on the molecular basis for the heterogeneity of voltage-gated potassium (Kv) channels in normal and in diseased hearts, and the mechanisms/sites of actions of currently available Kv channel modulators that may provide therapeutic benefits for problems in the heart and elsewhere. Our focus here is the slow delayed rectifier (IKs) channel, an important determinant of action potential duration in human heart. The IKs channel consists of at least two components: KCNQ1 channel and KCNE1 auxiliary subunit. Mutations in kcnq1 & kcne1 genes have been linked to abnormalities in cardiac repolarization and increased risk for arrhythmias (long & short QT syndromes, LQT & SQT, and familial atrial fibrillation, fAF). Recent data from our lab and from others have suggested that the subunit composition of cardiac IKs channels may be more complex than previously believed. Transcripts of other members of the KCNE family (KCNE2 - KCNE5) have been detected in human heart, and in heterologous expression systems these KCNE subunits can all associate with the KCNQ1 channel to confer distinct channel phenotypes. We are particularly interested in KCNE2, because we have confirmed the presence of KCNE2 protein in human heart, and mutations in the kcne2 gene have been linked to LQT6 or fAF. We have shown that in heterologous expression systems KCNE2 can associate with the IKs (KCNQ1/KCNE1) channel complex to reduce its current amplitude without changing its gating kinetics. Furthermore, our preliminary pulse-chase experiments suggest that the partnership between KCNQ1 and KCNE subunits is not permanent: KCNE subunits can dissociate from KCNQ1/KCNE complexes to be replaced by new ones. These observations suggest the intriguing possibility that the subunit composition of cardiac IKs channels is dynamic: KCNE1 functions as the major auxiliary subunit to set the IKs gating kinetics, while KCNE2 functions as a dynamic regulator to fine tune the IKs current amplitude. In this proposal, we will seek direct evidence for the role of native KCNE2 in cardiac IKs channel function. We also want to quantify the relationship between KCNE1 & KCNE2 expression levels, their KCNQ1 binding affinity, and the IKs subunit composition (Aim 1), to apply the above information to the study of mechanisms for IKs remodeling in aging hearts (Aim 2), and to determine the structural basis for the dynamic interactions between KCNQ1 and the two KCNE subunits (Aim 3). To achieve these Aims, we will use a multidisciplinary approach of electrophysiology, molecular biology, protein biochemistry, confocal microscopy and molecular modeling. Importantly, we will study not only channels expressed in heterologous systems but also native channels in cardiac myocytes. PUBLIC HEALTH RELEVANCE: Our data will provide novel insights into the dynamic nature of cardiac IKs channel subunit composition. We believe this is one of the mechanisms by which cardiac myocytes fine tune the IKs amplitude in response to stress. We will apply this knowledge to the study of IKs remodeling during physiological and pathological aging. Finally, we will obtain structural information on IKs channel subunit interactions, and use this information to refine 3-D models of the IKs channel in different gating states. These models will be useful in structure-based design of IKs activators that can combat acquired & congenital LQT syndromes.

描述（由申请人提供）：本研究项目的长期目标是提供关于正常和患病心脏中电压门控钾（Kv）通道异质性的分子基础的信息，以及目前可用的Kv通道调节剂的作用机制/位点，这些调节剂可能为心脏和其他部位的问题提供治疗益处。我们的重点是慢延迟整流（IKs）通道，一个重要的决定因素的动作电位持续时间在人类心脏。IKs通道至少由两个部分组成：KCNQ 1通道和KCNE 1辅助亚基。kcnq 1和kcne 1基因突变与心脏复极异常和心律失常风险增加（长QT综合征和短QT综合征，LQT和SQT，以及家族性房颤，fAF）有关。我们实验室和其他实验室的最新数据表明，心脏IKs通道的亚基组成可能比以前认为的更复杂。KCNE家族的其他成员（KCNE 2-KCNE 5）的转录本已在人类心脏中检测到，并且在异源表达系统中，这些KCNE亚基都可以与KCNQ 1通道结合以赋予不同的通道表型。我们对KCNE 2特别感兴趣，因为我们已经证实了KCNE 2蛋白在人类心脏中的存在，并且kcne 2基因的突变与LQT 6或fAF有关。我们已经证明，在异源表达系统中，KCNE 2可以与IKs（KCNQ 1/KCNE 1）通道复合物结合，以降低其电流幅度，而不改变其门控动力学。此外，我们初步的脉冲追踪实验表明，KCNQ 1和KCNE亚基之间的伙伴关系不是永久的：KCNE亚基可以从KCNQ 1/KCNE复合物中解离出来，被新的亚基取代。这些观察结果表明，心脏IKs通道的亚基组成是动态的有趣的可能性：KCNE 1作为主要的辅助亚基设置IKs门控动力学，而KCNE 2作为动态调节器微调IKs电流幅度。在这个提议中，我们将寻找天然KCNE 2在心脏IKs通道功能中作用的直接证据。我们还希望量化KCNE 1和KCNE 2表达水平、KCNQ 1结合亲和力和IKs亚基组成之间的关系（目的1），将上述信息应用于衰老心脏IKs重构机制的研究（目的2），并确定KCNQ 1和两个KCNE亚基之间动态相互作用的结构基础（目的3）。为了实现这些目标，我们将使用电生理学，分子生物学，蛋白质生物化学，共聚焦显微镜和分子建模的多学科方法。重要的是，我们不仅研究在异源系统中表达的通道，而且研究心肌细胞中的天然通道。公共卫生相关性：我们的数据将为心脏IKs通道亚基组成的动态性质提供新的见解。我们认为这是心肌细胞在应激反应中微调IKs振幅的机制之一。我们将把这些知识应用于生理和病理衰老过程中IKs重构的研究。最后，我们将获得IKs通道亚基相互作用的结构信息，并使用这些信息来细化不同门控状态下IKs通道的3D模型。这些模型将是有用的IKs激活剂，可以打击后天性和先天性LQT综合征的结构为基础的设计。