MOLECULAR BASIS FOR Kv CHANNEL HETEROGENEITY IN THE HEART

描述(申请人提供)：这项研究项目的长期目标是提供关于正常和疾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/部位，这些调节剂可能为心脏和其他地方的问题提供治疗益处。这里我们关注的是慢延迟整流(IKS)通道，它是人体心脏动作电位时程的重要决定因素。IKS信道至少由两个部分组成：KCNQ1信道和KCNE1辅助子单元。KCNQ1和KCNE1基因突变与心脏复极异常和心律失常(长和短QT综合征，LQT和SQT，以及家族性心房颤动，Faf)的风险增加有关。来自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之前认为的更复杂。KCNE家族其他成员(KCNE2-KCNE5)的转录本已经在人类心脏中被检测到，在异源表达系统中，这些KCNE亚基都可以与KCNQ1通道结合，从而赋予不同的通道表型。我们对KCNE2特别感兴趣，因为我们已经证实在人类心脏中存在KCNE2蛋白，并且kcne2基因的突变已经与LQT6或Faf连锁。我们已经证明，在异源表达系统中，KCNE2可以与IKS(KCNQ1/KCNE1)通道复合体结合，在不改变其门控动力学的情况下降低其电流幅度。此外，我们的初步脉冲追逐实验表明，KCNQ1和KCNE亚基之间的伙伴关系不是永久性的：KCNE亚基可以从KCNQ1/KCNE复合体中解离出来，被新的复合体取代。这些观察结果提示了一种有趣的可能性，即心脏IKS通道的亚基组成是动态的：KCNE1作为主要的辅助亚基来设置IKS门控动力学，而KCNE2作为动态调节器来微调IKS电流幅度。在这项提案中，我们将寻求天然KCNE2在心脏IKs通道功能中所起作用的直接证据。我们还想量化KCNE1和KCNE2的表达水平、它们的KCNQ1结合亲和力和IKS亚基组成(目标1)之间的关系，将上述信息应用于老年心脏IKS重塑机制的研究(目标2)，并确定KCNQ1和这两个KCNE亚基之间动态相互作用的结构基础(目标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等多学科方法。重要的是，我们不仅将研究异种系统中表达的通道，还将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通道亚单位组成的动态性质提供新的见解。我们认为这是心肌细胞微调IKS幅度以应对压力的机制之一。我们将把这些知识应用于生理和病理老化过程中IKs重塑的研究。最后，我们将获得关于IKS通道亚单位相互作用的结构信息，并利用这些信息来提炼不同门控状态下IKS通道的三维模型。这些模型将有助于基于结构的IKS激活剂的设计，以对抗获得性和先天性LQT综合征。

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.