hERG Channel Assembly and Trafficking

描述（由申请人提供）：这项工作的长期目标是揭示遗传性和获得性长QT综合征（LQT）的决定因素，并取得进展，最终允许开发更多药物，同时保护那些有心脏性猝死风险的人。这项研究的重点是组成和功能分析的hERG 1a和1b亚基有助于通道的基础心脏IKr，一个关键的目标，遗传性和获得性LQT。当前项目的具体目标是（1）阐明天然组织中同源和异源hERG通道的发育和性别特异性特征，（2）解析由异源系统中的一级序列确定的这些亚基的化学计量，以及天然系统中发现的化学计量，（3）理解先前定义的同源和异源通道药理学差异的生物物理学基础，和（4）表征最新定义的hERG 1b同种型特异性疾病的机制。该提案的一个高影响力组成部分是使用来自人类供体的大量正常心室组织样本，以及对个体标本进行配对分析，以了解通道组成和对导致获得性长QT综合征（LQT）的药物挑战的生理反应。两个独立的，创新的策略将用于确定在异源和天然组织的亚基化学计量。药物对同聚体和异聚体通道的差异效力的潜在机制将通过测试与1a和1b亚基的结构和功能差异相关的两个假设来确定。1b亚基的扰动如何导致疾病将通过实验进行评估，这些实验报告了源自经证实的基因组筛选方法的新型突变引起的细胞运输或功能改变的变化，通过评估与LQT人群持续相关的遗传修饰剂，以及通过选择性敲低天然组织中的1b亚基。这些实验有望阐明hERG 1a和1b亚基如何促进天然IKr，以及它们的不同表达谱如何决定儿童、男性和女性的正常功能和对长QT综合征和心源性猝死的易感性。

DESCRIPTION (provided by applicant): The long-term goals of this work are to uncover determinants of inherited and acquired long QT syndrome (LQT), and to make advances that will ultimately allow more drugs to be developed while protecting those at risk from sudden cardiac death. This study focuses on composition and functional analysis of hERG 1a and 1b subunits contributing to channels underlying cardiac IKr, a critical target for inherited and acquired LQT. The specific aims of the current project are to (1) elucidate the developmental and sex-specific profiles of homomeric and heteromeric hERG channels in native tissues, (2) resolve the stoichiometry of these subunits determined by primary sequence in heterologous systems, and that found in native systems, (3) understand the biophysical basis for pharmacological differences previously defined for homomeric and heteromeric channels, and (4) characterize mechanisms of disease specific to the most recently defined hERG 1b isoform. A high-impact component of the proposal is the use of a large sample of normal ventricular tissue from human donors, and the paired analysis of individual specimens with respect to channel composition and physiological response to challenge by drugs that cause acquired long QT syndrome (LQT). Two independent, innovative strategies will be utilized for determining subunit stoichiometry in both heterologous and native tissues. The mechanism underlying differential potency of drugs to homomeric and heteromeric channels will be determined by testing two hypotheses relating to structural and functional differences of the 1a and 1b subunits. How perturbation of the 1b subunit contributes to disease will be evaluated with experiments reporting changes in cellular trafficking or functional alterations caused by novel mutations derived from a proven genomic screening approach, by the evaluation of a genetic modifier persistently associated with LQT populations, and by selective knockdown of the 1b subunit in native tissues. These experiments are expected to elucidate how hERG 1a and 1b subunits contribute to native IKr and how their different expression profiles dictate normal function and susceptibility to long QT syndrome and sudden cardiac death in children, men and women.