STRUCTURE-FUNCTION RELATION & MODULATION OF Kv CHANNELS

描述（由申请人提供）：该项目具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）了解为什么Kv通道成分的突变导致功能丧失或获得，以及（3）确定针对心脏Kv通道的新治疗策略。该提案的重点是慢速延迟整流器（IK）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，IK 起到“复极储备”的作用：响应 β 肾上腺素能刺激，IK 增加其电流幅度，以防止动作电位持续时间 (APD) 过度延长。在人类心房中，IK 可能是心房颤动 (AF) 的一个责任因素。已鉴定出八种与家族性房颤相关的“功能获得”KCNQ1 突变。更重要的是，据报道，由于瓣膜疾病导致的获得性房颤，KCNE1 上调，这表明在这些情况下 IK 增加，可能有助于 APD 缩短和房颤持续。我们已经证明，在人类心脏中表达的另一个 KCNE 亚基 KCNE2 与成人心肌细胞中的 KCNQ1 和 KCNE1 共定位。它可以与IKs通道结合形成KCNQ1/KCNE1/KCNE2三元复合物。 KCNE2 降低了 IKs 电流幅度而不影响其门控动力学。家族性 AF 相关突变 R27C 的鉴定突显了 KCNE2 作为 IK 调节剂的重要性，该突变否定了 KCNE2 目前对 IK 的抑制作用。 KCNE1 和 KCNE2 对心脏 IK 电流幅度的调节之间的关系尚不清楚。它们对 KCNQ1 通道功能产生明显不同影响的机制也不是。该项目旨在解决这些问题。我们是 3 个具有互补专业知识的研究小组（Tseng - 通道生物物理学、Cui - 计算模型和 Tian - 核磁共振），共同努力实现以下具体目标。目标1 目的是确定 KCNQ1 通道中跨膜螺旋 (TMH) 的堆积和门控相关运动。目标 2 是确定 KCNE1 关联对 KCNQ1 通道中 TMH 相互作用以及 KCNE1 和 KCNQ1 之间接触的影响。目标 3 是确定 KCNE2 和 KCNQ1 之间的接触以及这些接触的状态依赖性。这些实验中确定的空间关系将用于约束 KCNQ1 同源模型处于开放和封闭状态。我们还将精炼后的 KCNE NMR 结构以与实验数据一致的方式对接至 KCNQ1 同源模型。最后，我们将测试膜渗透性 KCNE2 模拟肽是否可以破坏 KCNQ1/KCNE2 相互作用并增加心肌细胞中的 IK 电流幅度（目标 4）。这可以深入了解 KCNE1 和 KCNE2 在 IK 幅度调节方面的关系。它还可作为针对 KCNQ1/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.