Structural rationale for open-state-inducing mutation in human Iks-producing potassium channel complex

 描述(申请人提供)：人钾通道KCNQ1是一种多面性的螺旋膜蛋白，负责动作电位后心肌细胞的复极化以及几种上皮组织中的水和盐的动态平衡。KCNQ1功能障碍会导致各种疾病，其中最常见的与心脏病有关。在心脏，KCNQ1与辅助蛋白KCNE1结合，形成主要负责动作电位持续时间的钾通道。KCNQ1和/或KCNE1功能丧失突变具有严重的表型，导致听力丧失和长QT综合征。功能获得突变与婴儿猝死综合征、家族性心房颤动和短QT综合征有关。KCNQ1中的一个这样的突变是KCNQ1中的V141M，它完全依赖于KCNQ1与KCNE1的关联，被认为是三维结构裂隙的一部分，其中有几个功能获得突变。KCNQ1和KCNE1中的一些致病多态是跨膜区内的单一氨基酸突变。尽管存在KCNQ1的几个同源模型，但这些模型不能解释为高分辨率；这种低置信度是由于对KCNQ1(电压敏感域；Q1-VSD)具有电压敏感性的四个螺旋束跨膜结构域与先前确定的同源结构(~20%)之间的序列同源性较低。利用V141M和已知的锁定Q1-VSD(E160R-Q234E)突变，本项目的目标是建立一个可靠的、实验限制的、原子分辨的Q1-VSD：KCNE1复合体的开放状态结构，用于解释包括V141M在内的心房颤动诱导突变家族的病因学。本研究包括三个部分：1)确定开放状态Q1-VSD的结构；2)在有和没有V141M突变的情况下，在KCNE1和Q1-VSD之间产生体外约束；3)利用电生理学产生结构-功能约束，最终应用建模软件Rosetta中的所有约束将电压传感器结构域对接到KCNE1。开发一个实验受限的KCNQ1跨膜结构域的原子分辨模型，因为它与KCNE1络合，有望阐明与这种络合物相关的许多致病突变的结构基础。

 DESCRIPTION (provided by applicant): The human potassium channel KCNQ1 is a polytopic, helical membrane protein responsible for repolarization of heart muscle cells after the action potential, as well as water and salt homeostasis in several epithelial tissues. Malfunctioning KCNQ1 leads to various diseases, the most common of which are associated with heart disease. In the heart, KCNQ1 associates with the accessory protein KCNE1 to form the potassium channel largely responsible for the duration of the action potential. KCNQ1 and/or KCNE1 loss-of-function mutations have severe phenotypes resulting in loss of hearing and long QT syndrome. Gain-of-function mutations have been linked to sudden infant death syndrome (SIDS), familial atrial fibrillation and short QT syndrome. One such mutation that has been characterized in vivo, V141M in KCNQ1, is completely dependent on KCNQ1 association with KCNE1 and is thought to be a part of a cleft in the three-dimensional structure, where several gain-of-function mutations are located. Several disease-inducing polymorphisms within KCNQ1 and KCNE1 are single amino acid mutations within the transmembrane region. Although several homology models of KCNQ1 exist, these models are not interpretable to high resolution; this low degree of confidence is due to the low sequence identity between the four helical bundle transmembrane domain that confers voltage sensitivity to KCNQ1 (voltage-sensor domain; Q1-VSD) and previously determined, homologous structures (~20%). Utilizing the V141M and the known lock-on Q1-VSD (E160R-Q234E) mutations, the goal of this project is to build a reliable, experimentally-restrained, atomic-resolution structure of the open state of Q1-VSD:KCNE1 complex, which will be used to explain the etiology of the family of atrial fibrillation- inducing mutations, including V141M. The proposed research consists of three parts: 1) determine the structure of the open state Q1-VSD, 2) generate in vitro restraints between KCNE1 and Q1-VSD with and without the V141M mutation, and 3) generate structure-function restraints using electrophysiology, ultimately applying all of the restraints in the modeling software Rosetta to dock the voltage sensor domain to KCNE1. Developing an experimentally-constrained atomic-resolution model of the transmembrane domain of KCNQ1 as it complexes with KCNE1 promises to elucidate the structural underpinnings of the many disease-inducing mutations associated with this complex.