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

项目摘要 心脏节律由整个心脏的同步电脉冲触发和维持。缓慢 延迟整流钾电流（IKs）对心脏动作电位的适当复极化有重要作用， 因此对于维持健康的心律是必要的。IKs的分子相关性被确定为 一种离子通道复合物，由两种膜蛋白组成：KCNQ 1和KCNE 1。KCNQ 1（也称为 作为Kv7.1或KvLQT 1）是IKs通道复合物的成孔亚基。它属于电压门控 钾通道超家族单独表达KCNQ 1可产生快速激活和失活的 延迟整流钾电流，其性质，然而，不匹配的心脏IKs。 KCNQ 1必须与其辅助亚基KCNE 1共组装以产生IKs电流。KCNE 1是一个小型 一种单跨膜蛋白，通过减慢KCNQ 1的生物物理特性， 激活和失活动力学，通过改变通道开放概率的电压依赖性， 增加了单沟道电导。尽管在过去的十年里对IKs进行了深入的研究，但它仍然存在 KCNE 1如何在分子水平上调节和改变KCNQ 1的功能还很不清楚。另一方面，在一项研究中， 由于kcnq 1或kcne 1基因在心脏功能中的重要作用，其突变可导致多种心脏疾病。 如家族性心房颤动、长QT综合征、短QT综合征，甚至婴儿猝死。 然而，作为一个潜在的药物靶点，小分子如何潜在地 操纵KCNQ 1/KCNE 1通道复合物的功能在很大程度上是未知的。在此我提议 通过实现三个直接目标，对KCNQ 1/KCNE 1进行系统的基于结构的研究：1） KCNQ 1/KCNE 1通道复合物的结构和生物化学表征; 2）高通量小 使用基于蛋白脂质体的通量测定法进行分子筛选; 3）结构和功能阐明 KCNQ 1/KCNE 1复合物与小分子之间的相互作用。在K99指导阶段，博士。 罗德里克麦金农的实验室，我将进行单粒子冷冻电镜研究，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测定法， 可以进行高通量小分子筛选以寻找靶向 KCNQ 1/KCNE 1通道复合物。在R 00独立阶段，大规模的小分子筛选将在 完成，有希望的命中将使用生物化学和生物物理方法，如细胞， 基于膜片钳分析。最后，IKs通道复合物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 研究单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.