K+ Channel Trafficking and Modulation by Mink and MiRP1

描述(申请人提供)：电压门控钾(Kv)通道使可兴奋的细胞(如心肌细胞)重新极化。心肌细胞Kv通道功能障碍会导致危及生命的心律失常，但这些通道也是有用的抗心律失常药物靶点。因此，了解它们的功能、调节和分子组成，并确定它们在地区和物种之间的差异是至关重要的。目前的建议借鉴了我们过去十年在克隆和确定KCNE家族成员的不同生理作用方面的工作，这些成员是单跨膜结构域Kv通道辅助亚单位。在我们之前发现KCNE2突变与遗传性和获得性人类室性心律失常相关之后，最近我们建立了Kcne2(-/-)小鼠系，并用它来确定KCNE2在成年小鼠心室中的主要作用-调节两个Kv通道及其天然电流相关性：Kv4.2(Ito，f)和出人意料的Kv1.5(Ik，Slow1)。我们还定义了KCNE1作为KCNQ1a亚基的内吞伴侣的新角色，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突变与房性和室性心律失常相关。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。大多数类型的房颤没有已知的遗传基础，并与其他因素相关，如年龄，或心脏或肺手术后。更充分地了解所有这些Kv亚基的天然生理学，以及它们如何在遗传性、年龄起病或手术后(获得性)形式的房颤中发挥作用，对于改善人类心脏健康非常重要。在这里，我们建议利用Kcne2(-/-)小鼠(表现为起搏诱发的房颤)、术后房颤的兔和猪模型、人心房组织的验证性实验以及硅胶多尺度心房模型，来确定KCNE2在心房生理学和房颤病因中的作用。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调节哪些心房Kv复合体，它的遗传破坏如何导致房颤和Kv通道重构，这些机制如何反映较大动物手术后的房颤，以及Sp1，miR-1和miR-133在这种重构中的作用。其次，我们将使用电生理学/计算机模拟的方法来确定KCNE2在小鼠和兔心房中的功能，比较Kcne2基因突变和术后房颤对细胞功能的影响，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和间盘(ID)之间的关系，并确定为什么KCNE2的中断阻止了Kv1.5 ID靶向在小鼠脑室而不是心房。 与公共健康相关：特定的钾通道控制心脏复极，以及时结束每一次心跳；编码钾通道的基因中的遗传基因变异，包括KCNQ1、KCNE1和KCNE2，会导致人类致命的心律失常。在美国，250万人遭受着房颤的折磨，这些基因的突变可能会导致房颤，但更常见的是与衰老和一些外科手术有关。我们的建议旨在确定钾通道在心房中的机制作用，主要集中在KCNE2，并揭示导致KCNE2功能障碍的分子事件在遗传性和获得性房颤中，以便于未来的抗心律失常治疗和预防策略。

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.