K+ Channel Trafficking and Modulation by Mink and MiRP1

描述（由申请人提供）：电压门控钾（Kv）通道使可兴奋细胞（如心肌细胞）再极化。心肌细胞Kv通道功能障碍可导致危及生命的心律失常，但这些通道也是抗心律失常药物的有效靶点。因此，有必要了解它们的功能、调控和分子组成，并确定它们在区域和物种之间的差异。目前的建议借鉴了我们在克隆和定义KCNE家族成员的单一跨膜域Kv通道辅助亚基的不同生理作用方面的前十年工作。根据我们之前的发现，KCNE2突变与遗传性和获得性人类室性心律失常有关，最近我们建立了KCNE2（-/-）小鼠系，并使用它来确定KCNE2在成年小鼠心室中的主要作用-两个Kv通道及其原生电流相关的调制：Kv4.2 （Ito,f）和Kv1.5 （IK,slow1）。我们还定义了KCNE1的新作用，作为KCNQ1 a亚基的内吞伴侣，并发现KCNE1和KCNE2都可以影响功能性Kv通道的a亚基组成。KCNQ1、KCNE1和KCNE2突变与心房和室性心律失常都有关。Kv1.5突变与心房颤动（AF）有关，其功能在人类心脏中相对具有心房特异性，可能使其成为心房抗心律失常药物的有用靶点。大多数形式的房颤没有已知的遗传基础，并与其他因素有关，如年龄，或心脏或肺部手术后。更全面地了解所有这些Kv亚基的天然生理学，以及它们如何导致遗传性、年龄性或术后（获得性）形式的房颤，对改善人类心脏健康非常重要。在这里，我们建议利用KCNE2（-/-）小鼠（表现起搏性房颤）、兔和猪术后房颤模型、人心房组织验证实验和多尺度心房模型来确定KCNE2在心房生理学和房颤病因学中的作用。这些研究包括三个具体目标。首先，我们将使用分子方法来确定KCNE2调节的心房Kv复合物，其遗传破坏如何导致房颤和Kv通道重塑，这些机制如何反映大型动物术后房颤，以及Sp1， miR-1和miR-133在这种重塑中的作用。其次，我们将使用电生理学/计算机建模方法来确定KCNE2在小鼠和兔心房中的功能，比较KCNE2基因破坏和术后房颤引起的细胞功能影响，并从细胞到组织水平模拟由此产生的心律失常的机制基础。第三，我们将定义KCNE2、Kv1.5和间插椎间盘（IDs）之间的关系，并确定为什么KCNE2破坏会阻止Kv1.5 ID靶向小鼠心室而不是心房。

DESCRIPTION (provided by applicant): Voltage-gated potassium (Kv) channels repolarize excitable cells such as cardiac myocytes. Dysfunction of cardiac myocyte Kv channels causes life-threatening cardiac arrhythmias, but these channels are also useful antiarrhythmic drug targets. Thus, it is essential to understand their function, regulation and molecular composition, and determine how these differ regionally and between species. The current proposal draws from our preceding decade of work on cloning and defining the diverse physiological roles of members of the KCNE family of single- transmembrane-domain Kv channel ancillary subunits. Following our previous findings that KCNE2 mutations associate with inherited and acquired human ventricular arrhythmias, more recently we generated the kcne2 (-/-) mice line and used it to determine the primary roles of KCNE2 in adult murine ventricles - modulation of two Kv channels and their native current correlates: Kv4.2 (Ito,f) and, unexpectedly, Kv1.5 (IK,slow1). We also defined a new role for KCNE1, as an endocytic chaperone of the KCNQ1 a subunit, and found that both KCNE1 and KCNE2 can influence the a subunit composition of functional Kv channels. KCNQ1, KCNE1 and KCNE2 mutations associate with both atrial and ventricular arrhythmias. Kv1.5 mutations associate with atrial fibrillation (AF), and its function is relatively atrial-specific in human heart, potentially making it a useful target for atrial antiarrhythmics. Most forms of AF have no know genetic basis, and correlate with other factors such as aging, or following surgery to the heart or lungs. A fuller understanding of the native physiology of all these Kv subunits, and how they contribute to both inherited, and age-onset or post-surgery (acquired) forms of AF, is important to improving human cardiac health. Here, we propose to determine the roles of KCNE2 in atrial physiology and in the etiology of AF, utilizing kcne2 (-/-) mice (which exhibit pacing-induced AF), rabbit and swine models of post-operative AF, confirmatory experiments with human atrial tissue, and in silico multiscale atrial models. The studies comprise three Specific Aims. First, we will use a molecular approach to determine which atrial Kv complexes KCNE2 regulates, how its genetic disruption causes AF and Kv channel remodeling, how these mechanisms mirror post-operative AF in larger animals, and the role of Sp1, miR-1 and miR-133 in this remodeling. Second, we will use an electrophysiology/computer modeling approach to determine the function of KCNE2 in mouse and rabbit atria, compare the cellular functional effects arising from kcne2 genetic disruption and post-operative AF, and simulate the mechanistic basis for the resultant arrhythmias, from the cellular to the tissue level. Third, we will define the relationship between KCNE2, Kv1.5, and the intercalated discs (IDs), and determine why KCNE2 disruption prevents Kv1.5 ID targeting in the murine ventricles but not atria. PUBLIC HEALTH RELEVANCE: Specific potassium channels govern cardiac repolarization to end each heart-beat in a timely fashion; inherited gene variants in the genes that encode potassium channels, including KCNQ1, KCNE1 and KCNE2, cause lethal cardiac arrhythmias in man. Atrial fibrillation, which afflicts 2.5 million people in the United States, can be caused by mutation in these genes but is more commonly associated with aging and some surgical procedures. Our proposal is designed to determine the mechanistic role of potassium channels in the atrium, focusing primarily on KCNE2, and uncover molecular events leading up to dysfunction of KCNE2 in inherited and acquired forms of atrial fibrillation, in order to facilitate future antiarrhythmic therapy and prevention strategies.