Pathogenesis of HERG Mutations in Human Long QT Syndrome

描述（由申请人提供）：先天性长 QT 综合征 (LQTS) 是一种 与心脏复极延迟和 QT 延长相关的疾病 心电图上的间隔，可能导致室性心律失常 伴有心源性猝死。 LQTS (LQT2) 的主要形式之一是由 编码人类 ether-a-go-go 相关基因 (HERG) 的突变 快速激活延迟整流钾通道。迄今为止，已有超过 100 LQTS 患者中已发现 HERG 突变。我们之前的工作 已经表明 LQT2 中 HERG 通道功能丧失的主要机制是 有缺陷的蛋白质运输，导致突变通道无法 到达细胞表面。我们还表明，高亲和力 HERG 通道阻断剂 可以纠正一些 LQT2 突变体的蛋白质运输缺陷。的目标 该提案是（1）研究缺陷蛋白质运输的机制 LQT2 突变通道，以及 (2) 确定 HERO 通道阻断剂如何拯救 运输有缺陷的 LQT2 突变通道。我们的假设是 (1) LQT2 突变导致 HERO 蛋白错误折叠或不正确组装 通过质量控制系统的认可，导致 ER 保留和降解 蛋白酶体，以及 (2) 以高亲和力作用与 HERO 通道结合的药物 作为药理学伴侣，促进正确折叠或组装 允许运输到质膜的构象。我们将测试 这些假设有四个具体目标： 目标 I 确定 LQT2 是否 突变导致突变通道的错误折叠或不当组装；目标 2 到 研究分子伴侣在 LQT2 突变体 ER 保留中的作用 渠道；目标 3 研究 LQT2 突变体的机制 被蛋白酶体识别并降解；目标 4 阐明 高亲和力 HERG 通道阻断剂纠正缺陷的机制 LQT2 突变通道的蛋白质运输。我们将使用以下组合 生物化学、免疫组织化学和膜片钳技术研究野生型 HERG 和 LQT2 突变通道在转染的组织培养细胞中表达 在无细胞系统中。这些研究将加强我们对如何 错误折叠和不正确组装的 LQT2 突变通道被识别， ER 质量控制系统保留和降解以及 HERG 如何通道 阻断剂可以修改这些过程并拯救 LQT2 突变通道。阐明 这些机制是朝着发展方向迈出的重要一步 先天性 LQTS 治疗的药理学策略。

DESCRIPTION (provided by applicant): Congenital long QT syndrome (LQTS) is a disease associated with delayed cardiac repolarization and prolonged QT intervals on the electrocardiogram, which can lead to ventricular arrhythmia with cardiac sudden death. One of the major forms of LQTS (LQT2) is caused by mutations in the human ether-a-go-go-related gene (HERG) that encodes the rapidly activating delayed rectifier potassium channel. To date, more than 100 HERG mutations have been identified in patients with LQTS. Our previous work has shown that a major mechanism for loss of HERG channel function in LQT2 is defective protein trafficking which results in failure of mutant channels to reach the cell surface. We also showed that high affinity HERG channel blockers can correct defective protein trafficking of some LQT2 mutants. The goals of this proposal are (1) to study the mechanisms of defective protein trafficking of LQT2 mutant channels, and (2) to determine how HERO channel blockers rescue trafficking defective LQT2 mutant channels. Our hypotheses are (1) LQT2 mutations cause misfolding or improper assembly of HERO protein which is recognized by quality control system leading to ER retention and degradation by the proteasome, and (2) drugs that bind to HERO channels with high affinity act as pharmacological chaperones to promote proper folding or assembly in a conformation that permits trafficking to the plasma membrane. We will test these hypotheses by four specific aims: aim I to determine whether LQT2 mutations cause misfolding or improper assembly of mutant channels; aim 2 to study the role of molecular chaperones in the ER retention of LQT2 mutant channels; aim 3 to investigate the mechanisms by which LQT2 mutants are recognized and degraded by the proteasome; and aim 4 to elucidate the mechanisms by which high affinity HERG channel blockers correct defective protein trafficking of LQT2 mutant channels. We will use a combination of biochemical, immunohistochemical and patch clamp techniques to study wild type HERG and LQT2 mutant channels expressed in transfected tissue culture cells and in cell-free systems. These studies will strengthen our knowledge of how misfolded and improperly assembled LQT2 mutant channels are recognized, retained and degraded by the ER quality control system and how HERG channel blockers modify these processes and rescue LQT2 mutant channels. Elucidating these mechanisms is an important step towards the development of pharmacological strategies for therapies of congenital LQTS.