Investigating KCNQ1 Mistrafficking in Long QT Syndrome

调查长 QT 综合征中 KCNQ1 的错误贩卖

• 批准号: 10678328
• 负责人: Katherine R Moster
• 金额: $ 3.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678328
• 关键词: Action Potentials; Binding; Biological Assay; Cardiac; Cardiac Myocytes; Cell Line; Cell membrane; Cell surface; Cells; Cessation of life; Characteristics; Classification; Color; Cystic Fibrosis; DNA; Defect; Disease; Dose; Electrocardiogram; Endoplasmic Reticulum; Evaluation; Exhibits; Flow Cytometry; Fluorescence-Activated Cell Sorting; Functional disorder; Genes; Genome; Goals; Heart Arrest; Heart Diseases; Image; Immunofluorescence Immunologic; Impairment; Individual; Induced Mutation; Lead; Left; Libraries; Long QT Syndrome; Mammalian Cell; Mediating; Membrane Proteins; Methods; Molecular; Molecular Bank; Molecular Chaperones; Mutation; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Population; Potassium; Potassium Channel; Proteins; Research; Retinitis Pigmentosa; Rhodopsin; Risk; Role; Romano-Ward Syndrome; Route; Severities; Sorting; Specificity; Structure; Surface; Symptoms; Testing; Variant; Voltage-Gated Potassium Channel; drug development; high throughput screening; improved; insight; loss of function; loss of function mutation; mutant; mutation screening; next generation sequencing; novel; pharmacologic; response; small molecule; trafficking; treatment choice; variant of unknown significance; voltage

Project Summary Long QT syndrome (LQTS) is a cardiac disorder characterized by the prolongation of the latter portion of the electrocardiogram trace (the QT interval) that increases risk of cardiac arrythmia, cardiac arrest, and sudden unexpected death. Approximately 1 in 2500 individuals suffer from the congenital form of LQTS, with 30-50% of cases being caused by mutations in the voltage gated potassium channel protein KCNQ1 (type 1 LQTS, or LQT1). Over 250 LQT1-associated mutations in KCNQ1 have been identified, but the impact of these mutations on the channel’s structure and function, and whether there are common mechanisms through which these mutations lead to KCNQ1 dysfunction in LQT1, is still unknown. The primary goal of this proposed project is to explore mistrafficking as a potential mechanism of KCNQ1 loss of function in long QT syndrome. Previous studies of LQT1-associated mutations in the KCNQ1 voltage sensing domain (VSD) found that the majority decreased KCNQ1 trafficking to the plasma membrane and destabilized the VSD. Additional studies have shown that mutations in KCNQ1 can lead to retention in the endoplasmic reticulum (ER) and increased proteasomal degradation. This has led to the hypothesis that mistrafficking is a common mechanism of protein dysfunction in LQT1. Mistrafficking has been identified as a disease mechanism in several other membrane-protein associated diseases, such as cystic fibrosis and retinitis pigmentosa, and in the case of cystic fibrosis, drugs have been developed to rescue mistrafficking and alleviate disease symptoms. This leads to the additional hypothesis that drugs that bind nascent KCNQ1 channels and increase their stability can increase the trafficking of KCNQ1. In line with these hypotheses, I propose two aims: 1) to classify mutations across KCNQ1 based on their impact on KCNQ1 trafficking, and 2) to develop a high throughput screening method to identify small molecules that increase cell surface trafficking. In Aim 1, I will classify the trafficking of all possible KCNQ1 variants with a fluorescence-activated cell sorting (FACS)-based deep mutational scanning method. This will allow me to determine whether the majority of LQT1-associated mutations cause mistrafficking and also provide information on variants of unknown significance (VUS) and other KCNQ1 variants. In Aim 2, I will utilize immunofluorescence and high content imaging to screen for small molecules that increase WT or mutant KCNQ1 trafficking. This will test the hypothesis that KCNQ1 mistrafficking is rescuable with small molecules. Together, the results of these aims will provide further insight into the molecular mechanisms behind KCNQ1 dysfunction in LQT1 and explore a possible route for developing novel treatments for LQT1.

项目摘要 长QT综合征（LQTS）是一种以QT间期延长为特征的心脏疾病 增加心律失常风险的心电图描记（QT间期）的一部分，心脏 突然死亡，突然意外死亡。大约每2500人中就有1人患有先天性 LQTS的形式，其中30-50%的病例是由电压门控钾突变引起的 通道蛋白KCNQ 1（1型LQTS或LQT 1）。KCNQ 1中有超过250个LQT 1相关突变 但是这些突变对通道结构和功能的影响，以及是否 这些突变导致LQT 1中KCNQ 1功能障碍的共同机制， 仍然未知。这个项目的主要目标是探索误传作为一种潜在的 长QT综合征中KCNQ 1功能丧失的机制 KCNQ 1电压敏感域（VSD）中LQT 1相关突变的既往研究 发现大多数减少KCNQ 1运输到质膜和不稳定的 室间隔缺损其他研究表明，KCNQ 1的突变可能导致其滞留在内质网中 内质网（ER）和蛋白酶体降解增加。这导致了一种假设， 错配是LQT 1蛋白功能障碍的常见机制。误传一直是 被鉴定为其他几种膜蛋白相关疾病的疾病机制，例如 囊性纤维化和色素性视网膜炎，在囊性纤维化的情况下，已经开发了药物， 抢救误服，缓解疾病症状。这导致了另一个假设，即药物 结合新生的KCNQ 1通道并增加其稳定性可以增加KCNQ 1的运输。 根据这些假设，我提出了两个目标：1）基于KCNQ 1对突变进行分类 研究它们对KCNQ 1贩运的影响，2）开发一种高通量筛选方法， 增加细胞表面运输的小分子。在目标1中，我将把所有贩运人口 可能的KCNQ 1变体，具有基于荧光激活细胞分选（FACS）的深度突变， 扫描法这将使我能够确定大多数LQT 1相关突变是否 导致误检，并提供关于未知意义的变体（VUS）和其他 KCNQ 1变体。在目标2中，我将利用免疫荧光和高内涵成像来筛选 增加WT或突变型KCNQ 1运输的小分子。这将检验假设， KCNQ 1的错误反应可以用小分子来挽救。总之，这些目标的结果将提供 进一步深入了解LQT 1中KCNQ 1功能障碍背后的分子机制，并探索 开发LQT 1新疗法的可能途径。