STRUCTURE-FUNCTION RELATION & MODULATION OF Kv CHANNELS

描述（由申请人提供）：该项目有三个相互关联的目标：（1）提供心脏主要电压门控钾（Kv）通道功能的结构信息，（2）了解Kv通道组分突变导致功能丧失或获得的原因，（3）确定靶向心脏Kv通道的新型治疗策略。该建议的重点是慢延迟整流（IKs）通道。IKs有2个主要组成部分：成孔KCNQ 1通道和辅助KCNE 1亚基。在人类心室中，IKs起着“复极储备”的作用：响应于β-肾上腺素能刺激，IKs增加其电流幅度，以防止动作电位时程（APD）过度延长。在人类心房中，IKs可能是心房颤动（AF）的易患因素。已发现8种与家族性房颤相关的“功能获得性”KCNQ 1突变。更重要的是，已报告瓣膜疾病所致获得性房颤的KCNE 1上调，表明在这些条件下IKs增加，可导致APD缩短和房颤持续。我们已经证明，另一个KCNE亚基表达在人类心脏，KCNE 2，是共定位与KCNQ 1和KCNE 1在成人心肌细胞。它可以与IKs通道结合形成KCNQ 1/KCNE 1/KCNE 2三元复合物。KCNE 2降低IKs电流幅度而不影响其门控动力学。KCNE 2作为IKs调节剂的重要性通过鉴定家族性AF相关突变R27 C而突出，R27 C否定了KCNE 2对IKs的电流抑制作用。KCNE 1和KCNE 2在调节心脏IKs电流幅度方面的关系尚不清楚。它们对KCNQ 1通道功能的明显不同影响的机制也不清楚。该项目旨在解决这些问题。我们是3个研究小组，具有互补的专业知识（曾通道生物物理学，崔计算建模，和田核磁共振）作出协调一致的努力，以实现以下具体目标。要求1 是为了确定包装和门控相关的跨膜螺旋（TMH）在KCNQ 1通道的运动。目的二是研究KCNE 1与KCNQ 1通道中TMH相互作用的影响，以及KCNE 1与KCNQ 1之间的联系。目的3是确定KCNE 2和KCNQ 1之间的联系以及这种联系的状态依赖性。在这些实验中确定的空间关系将用于约束KCNQ 1同源模型在开放和封闭状态。我们还将对接的KCNE NMR结构，细化后，KCNQ 1同源性模型的方式与实验数据一致。最后，我们将测试膜渗透性KCNE 2模拟肽是否可以破坏KCNQ 1/KCNE 2相互作用并增加心肌细胞中IKs电流幅度（目的4）。这可能为了解KCNE 1和KCNE 2在IKs幅度调节方面的关系提供了见解。它也是靶向KCNQ 1/KCNE相互作用的治疗肽的原型。

DESCRIPTION (provided by applicant): This project has three interrelated objectives: (1) to provide structural information for the function of major voltage-gated potassium (Kv) channels in the heart, (2) to understand why mutations in Kv channel components lead to loss- or gain-of-function, and (3) to identify novel therapeutic strategies targeting cardiac Kv channels. The focus of this proposal is the slow delayed rectifier (IKs) channel. IKs has 2 major components: pore-forming KCNQ1 channel and auxiliary KCNE1 subunits. In human ventricles, IKs functions as a 'repolarization reserve': in response to b-adrenergic stimulation IKs increases its current amplitude to prevent excessive prolongation of action potential duration (APD). In human atria, IKs may be a liability factor for atrial fibrillation (AF). Eight 'gain-of-function' KCNQ1 mutations have been identified that are linked to familial AF. More importantly, KCNE1 upregulation has been reported for acquired AF due to valvular diseases, suggesting an increase in IKs under these conditions that can contribute to APD shortening and AF perpetuation. We have shown that another KCNE subunit expressed in human heart, KCNE2, is colocalized with KCNQ1 & KCNE1 in adult cardiac myocytes. It can associate with the IKs channel to form a KCNQ1/KCNE1/KCNE2 ternary complex. KCNE2 reduces the IKs current amplitude without affecting its gating kinetics. The importance of KCNE2 as a IKs regulator is highlighted by the identification of a familial AF-related mutation, R27C that negates the current suppressing effect of KCNE2 on IKs. The relationship between KCNE1 & KCNE2 in terms of their regulation of the IKs current amplitude in the heart is not clear. Nor is the mechanism(s) underlying their distinctly different effects on the KCNQ1 channel function. This project is designed to address these issues. We are 3 research groups with complementary expertise (Tseng - channel biophysics, Cui - computational modeling, and Tian - NMR) making a concerted effort to accomplish the following Specific Aims. Aim 1 is to determine the packing and gating-associated movements of transmembrane helices (TMHs) in the KCNQ1 channel. Aim 2 is to determine the impact of KCNE1 association on the TMH interactions in the KCNQ1 channel, and the contacts between KCNE1 & KCNQ1. Aim 3 is to determine the contacts between KCNE2 & KCNQ1 and the state-dependence of such contacts. Spatial relationships determined in these experiments will be used to constrain KCNQ1 homology models in open & closed states. We will also dock the KCNE NMR structures, after refinement, to the KCNQ1 homology models in a manner consistent with experimental data. Finally, we will test whether membrane permeable KCNE2-mimetic peptides can disrupt KCNQ1/KCNE2 interactions and increase the IKs current amplitude in cardiac myocytes (Aim 4). This could provide insights into the relationship between KCNE1 & KCNE2 in terms of IKs amplitude regulation. It also serves as a prototype for therapeutic peptides targeting KCNQ1/KCNE interactions.