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)提供心脏中主要电压门控钾（Kv）通道功能的结构信息，(2)了解Kv通道成分突变导致功能丧失或获得的原因，以及(3)确定针对心脏Kv通道的新治疗策略。本方案的重点是慢延迟整流器（IKs）通道。IKs有2个主要组成部分：成孔KCNQ1通道和辅助KCNE1亚基。在人类心室中，IKs起着“复极储备”的作用：在对b-肾上腺素能刺激的反应中，IKs增加其电流振幅以防止动作电位持续时间（APD）的过度延长。在人类心房中，IKs可能是心房颤动（AF）的一个危险因素。8个“功能获得性”KCNQ1突变已被确定与家族性房颤有关。更重要的是，KCNE1上调已被报道为由瓣膜疾病引起的获得性房颤，这表明在这些条件下IKs的增加可能有助于APD缩短和房颤永久化。我们已经证明，在人类心脏中表达的另一个KCNE亚基KCNE2在成人心肌细胞中与KCNQ1和KCNE1共定位。它可以与IKs通道结合形成KCNQ1/KCNE1/KCNE2三元配合物。KCNE2在不影响其门控动力学的情况下降低了IKs的电流幅值。KCNE2作为IKs调控因子的重要性被一个家族性af相关突变R27C的发现所强调，该突变否定了KCNE2目前对IKs的抑制作用。KCNE1和KCNE2调节心脏ik电流振幅的关系尚不清楚。它们对KCNQ1通道功能的明显不同影响的机制也不清楚。本项目旨在解决这些问题。我们是三个具有互补专业知识的研究小组（Tseng -通道生物物理，Cui -计算建模，Tian -核磁共振），共同努力实现以下具体目标。目标1

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.