hERG Channel Assembly and Trafficking

描述(申请人提供)：这项工作的长期目标是发现遗传性和获得性长QT综合征(LQT)的决定因素，并取得进展，最终将允许开发更多的药物，同时保护那些面临心脏性猝死风险的人。本研究侧重于HERG 1a和1b亚基的组成和功能分析，HERG 1a和1b亚基是遗传性和获得性LQT的关键靶点，是心脏IKR的基础通道。本项目的具体目标是：(1)阐明同体和异体HERG通道在自然组织中的发育和性别特异性；(2)解决这些亚基的化学计量学问题，这些亚基由异源系统中的初级序列决定，以及在自然系统中发现；(3)了解先前定义的同体和异体通道的药理差异的生物物理基础；以及(4)表征最近定义的HERG 1b亚型特有的疾病机制。该建议的一个高影响部分是使用来自人类捐赠者的正常心室组织的大样本，并对单个样本进行配对分析，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。将利用两种独立的、创新的策略来确定异种和天然组织中的亚单位化学计量比。通过检验与1a和1b亚基的结构和功能差异有关的两个假说，将确定药物对同质和异质通道的不同效力的机制。1b亚基的扰动如何有助于疾病的评估将通过报告细胞运输的变化或由经证实的基因组筛选方法产生的新突变引起的功能变化的实验来评估，通过评估与LQT群体持续相关的遗传修饰物，以及通过选择性地敲除天然组织中的1b亚基。这些实验有望阐明Herg1a和1b亚基如何参与原生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综合征和心脏性猝死的易感性。 公共卫生相关性：如果成功，这些研究将扩大目前对遗传性心律失常和那些因阻断用于其他治疗靶点的药物而引起的心律失常的分子基础的理解。他们将通过提供儿童、妇女和男子心源性猝死的分层风险信息，促进更好的药物安全筛查方法的发展。

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. PUBLIC HEALTH RELEVANCE: If successful, these studies will expand current understanding of the molecular basis for inherited arrhythmias and those caused by block from drugs intended for other therapeutic targets. They will facilitate development of better drug safety screening approaches by providing information of stratified risk for sudden cardiac death among children, women and men.