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.

项目概要 长QT综合征（LQTS）是一种以QT间期延长为特征的心脏病。 心电图迹线（QT 间期）的一部分，会增加心律失常、心脏疾病的风险 被捕、意外死亡。大约每 2500 人中就有 1 人患有先天性 LQTS 形式，30-50% 的病例是由电压门控钾突变引起的 通道蛋白 KCNQ1（1 型 LQTS 或 LQT1）。 KCNQ1 中超过 250 个与 LQT1 相关的突变 已被确定，但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 LQT1 中 KCNQ1 功能障碍的共同机制是 仍然未知。该拟议项目的主要目标是探索非法贩运作为潜在的 长QT综合征中KCNQ1功能丧失的机制。 先前对 KCNQ1 电压传感域 (VSD) 中 LQT1 相关突变的研究 发现大多数减少了 KCNQ1 向质膜的运输并破坏了 室间隔缺损。其他研究表明，KCNQ1 的突变可导致内质中的滞留 网状结构（ER）和蛋白酶体降解增加。这导致了这样的假设： 错误运输是 LQT1 蛋白功能障碍的常见机制。拐卖现象已 被确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，对于囊性纤维化，已经开发出药物来治疗 拯救非法贩运并减轻疾病症状。这导致了另一个假设，即药物 结合新生 KCNQ1 通道并增加其稳定性可以增加 KCNQ1 的运输。 根据这些假设，我提出两个目标：1）基于 KCNQ1 对突变进行分类 其对 KCNQ1 贩运的影响，以及 2) 开发高通量筛选方法来识别 增加细胞表面运输的小分子。在目标 1 中，我将对所有贩运行为进行分类 可能的 KCNQ1 变异体具有基于荧光激活细胞分选 (FACS) 的深度突变 扫描方法。这将使我能够确定大多数 LQT1 相关突变是否 造成错误贩运，并提供有关意义不明的变体 (VUS) 和其他信息 KCNQ1 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 KCNQ1 运输的小分子。这将检验以下假设： KCNQ1 的错误贩运可以用小分子来挽救。这些目标的结果将共同提供 进一步深入了解LQT1中KCNQ1功能障碍背后的分子机制并探索 开发 LQT1 新型治疗方法的可能途径。