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

产生人 Iks 的钾通道复合物中开放态诱导突变的结构原理

• 批准号: 8834238
• 负责人: Brett M Kroncke
• 金额: $ 5.42万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-27 至 2016-06-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8834238
• 关键词: Action Potentials; Affect; Affinity; Amino Acids; Atrial Fibrillation; Binding; Biochemical; Cardiac Myocytes; Cleaved cell; Collaborations; Complement; Complement 3; Complex; Computer software; Data; Disease; Docking; Ear; Electrophysiology (science); Epithelial; Etiology; Family; Genetic Polymorphism; Goals; Heart; Heart Diseases; Homeostasis; Homology Modeling; Human; In Vitro; Individual; Induced Mutation; Ion Channel; Kidney; Laboratories; Light; Link; Long QT Syndrome; Membrane Proteins; Modeling; Mutation; Nuclear Magnetic Resonance; Organ; Phenotype; Potassium; Potassium Channel; Preparation; Proteins; Research; Resolution; Signal Transduction; Sodium Chloride; Solutions; Spin Labels; Stomach; Structural Models; Structure; Sudden infant death syndrome; Surface; Syndrome; Tissues; Titrations; Training; Transmembrane Domain; Update; Uterus; Water; Work; base; career; flexibility; gain of function mutation; hearing impairment; in vivo; insight; loss of function mutation; mutant; nanosecond; patch clamp; protein complex; public health relevance; research study; restraint; sensor; structural biology; three dimensional structure; voltage

 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.

 描述（由申请人提供）：人钾通道KCNQ1是一种多位螺旋膜蛋白，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稳态。KCNQ1功能障碍会导致各种疾病，其中最常见的是与心脏病有关。在心脏中，KCNQ1与辅助蛋白KCNE1结合形成钾通道，主要负责动作电位的持续时间。KCNQ1和/或KCNE1功能丧失突变具有导致听力丧失和长QT综合征的严重表型。功能获得性突变与婴儿猝死综合征（SIDS）、家族性房颤和短QT综合征有关。一个这样的突变，已在体内表征，V141M在KCNQ1，是完全依赖于KCNQ1与KCNE1协会，并被认为是一个裂缝的一部分，在三维结构，其中几个功能获得性突变的位置。KCNQ1和KCNE1中的几种致病多态性是跨膜区域内的单个氨基酸突变。尽管存在KCNQ1的几种同源性模型，但这些模型无法解释为高分辨率;这种低置信度是由于赋予KCNQ1电压敏感性的四螺旋束跨膜结构域（电压传感器结构域; Q1-VSD）与先前确定的同源结构（~20%）之间的低序列同一性。利用V141M和已知的锁定Q1-VSD（E160R-Q234E）突变，本项目的目标是建立Q1-VSD：KCNE1复合物的开放状态的可靠的、实验限制的、原子分辨率的结构，其将用于解释包括V141M在内的心房颤动诱导突变家族的病因。该研究包括三个部分：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）使用电生理学产生结构-功能约束，最终将所有约束应用于建模软件Rosetta中以将电压传感器域对接到KCNE1。开发一个实验约束的原子分辨率模型KCNQ1的跨膜结构域，因为它与KCNE1复合物的承诺，阐明与此复杂的许多疾病诱导突变的结构基础。