Structural and Pharmacological Study of the KCNQ1/KCNE1 Potassium Channel Complex

项目总结 整个心脏由同步的电脉冲触发和维持心率。慢的 延迟整流钾电流(IKS)对心脏动作电位的适当复极有重要作用， 因此，这对保持健康的心率是必不可少的。IKs的分子相关性被鉴定为 一种离子通道复合体，由两个完整的膜蛋白KCNQ1和KCNE1组成。KCNQ1(也称为 如Kv7.1或KvLQT1)是IKS通道复合体的成孔亚基。它属于电压门控型 钾通道超家族。KCNQ1单独表达可产生快速激活和失活 延迟整流钾电流，然而，其特性与心脏iKs的特性不匹配。 KCNQ1必须与其辅助亚基KCNE1共同组装才能产生Iks电流。KCNE1是一家小型 通过减缓KCNQ1的生物物理性质而深刻改变其生物物理性质的单跨膜蛋白 激活和去激活动力学，通过改变通道开放概率的电压依赖性，以及通过 增加单通道电导。尽管在过去的十年里对iks进行了深入的研究，但它仍然 KCNE1是如何在分子水平上调节和改变KCNQ1的功能的，目前尚不清楚。另一方面， 由于其在心脏功能中的重要作用，KCNQ1或KCNE1基因突变可导致多种心脏疾病 如家族性房颤、长QT综合征、短QT综合征，甚至婴儿猝死。 然而，作为潜在的药物靶点，潜在的小分子潜在的分子决定因素 操纵KCNQ1/KCNE1通道复合体的功能在很大程度上是未知的。在这里，我建议携带 通过实现三个近期目标，对KCNQ1/KCNE1进行系统的结构调查：1) KCNQ1/KCNE1通道复合体的结构和生化特性；2)高通量小分子 用基于蛋白脂质体的通量分析进行分子筛选；3)结构和功能的鉴定 KCNQ1/KCNE1复合体与小分子的相互作用。在博士的K99指导阶段中。 罗德里克·麦金农的实验室，我将进行单粒子低温电磁研究，以确定高分辨率 IKS通道复合体的结构。同时，我将建立一种基于蛋白脂质体的通量分析，通过 可以进行哪些高通量小分子筛选来搜索靶向于 KCNQ1/KCNE1通道复合体。在R00独立阶段，将进行大规模的小分子筛分 完成，有希望的命中将使用生化和生物物理方法来表征，例如细胞- 基于膜片钳的分析。最后，iks通道复合体之间相互作用的分子细节 小分子将通过生物物理和生物化学方法进行研究。我的研究将揭开 IKs的分子性质，为基于结构的药物设计提供了蓝图，并作为一种范例 研究了单个跨膜辅助亚基和小分子对离子通道的调节。

PROJECT SUMMARY Heart rhythm is triggered and maintained by synchronized electrical impulses throughout the heart. The slow delayed rectifier potassium current (IKs) vitally contributes to proper repolarization of cardiac action potentials, and is thus essential for maintaining a healthy heart rhythm. The molecular correlate of IKs was identified to be an ion channel complex, formed by two integral membrane proteins: KCNQ1 and KCNE1. KCNQ1 (also known as Kv7.1 or KvLQT1) is the pore-forming subunit of the IKs channel complex. It belongs to the voltage-gated potassium channel superfamily. Expression of KCNQ1 alone generates a rapidly activating and inactivating delayed-rectifier potassium current, whose properties, however, do not match with those of the cardiac IKs. KCNQ1 must co-assemble with its ancillary subunit, KCNE1 to produce the IKs current. KCNE1 is a small single-transmembrane protein that profoundly modifies the biophysical properties of KCNQ1 by slowing activation and deactivation kinetics, by shifting the voltage-dependence of channel open probability, and by increasing the single channel conductance. Despite intensive studies on IKs in the past decade it remains largely unclear how KCNE1 modulates and alters the function of KCNQ1 at molecular level. On the other hand, due to its vital role in cardiac function, mutations in kcnq1 or kcne1 gene can lead to several cardiac diseases such as familial atrial fibrillation, long-QT syndromes, short-QT syndromes and even sudden death in infants. Yet, as a potential drug target, molecular determinants underlying how small molecules could potentially manipulate the function of KCNQ1/KCNE1 channel complexes are largely unknown. Here I propose to carry out systematic structure-based investigations on KCNQ1/KCNE1 by achieving three immediate goals: 1) Structural and biochemical characterization of the KCNQ1/KCNE1 channel complex; 2) High-throughput small molecule screen using a proteoliposome-based flux assay; 3) Structural and functional elucidation of the interaction between the KCNQ1/KCNE1 complex and small molecules. During the K99 mentored phase in Dr. Roderick MacKinnon's laboratory, I will carry out single particle cryo-EM study to determine the high-resolution structure of the IKs channel complex. At the same time, I will establish a proteoliposome-based flux assay, by which high-throughput small molecule screens can be carried out to search for compounds targeting the KCNQ1/KCNE1 channel complex. In the R00 independent phase, large-scale small molecule screens will be done, and promising hits will be characterized using both biochemical and biophysical methods such as cell- based patch clamp assays. Finally, molecular details underlying interactions between the IKs channel complex and small molecules will be investigated by biophysical and biochemical approaches. My research will unveil the molecular nature of IKs, provide a blueprint for structure-based drug design, and serve as a paradigm for studying ion channel modulation by single transmembrane accessory subunits and small molecules.