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间期延长相关的疾病 心电图上的间隔，这可能导致室性心律失常 心脏性猝死LQTS（LQT 2）的主要形式之一是由以下原因引起的 人类ether-a-go-go相关基因（HERG）中的突变，该基因编码 快速激活延迟整流钾通道。迄今为止，超过100 已在LQTS患者中发现HERG突变。我们以前的工作 已经表明LQT 2中HERG通道功能丧失的主要机制是 蛋白质运输缺陷，导致突变通道无法 到达细胞表面。我们还发现，高亲和力HERG通道阻断剂 可以纠正一些LQT 2突变体的蛋白质运输缺陷。的目标 这一建议是（1）研究缺陷蛋白质运输的机制 LQT 2突变通道，以及（2）确定HERO通道阻滞剂如何拯救 运输缺陷型LQT 2突变体通道。我们的假设是：（1）LQT 2 突变导致HERO蛋白的错误折叠或不正确组装， 质量控制系统认可，导致ER保留和降解， 蛋白酶体，和（2）以高亲和力结合HERO通道的药物起作用 作为药理学分子伴侣，以促进细胞内的正确折叠或组装， 允许运输到质膜的构象。我们将测试 这些假设通过四个具体目标：目标I，以确定是否LQT 2 突变导致突变通道的错误折叠或不正确组装;目的2 研究分子伴侣在LQT 2突变体内质网滞留中的作用 目的3：研究LQT 2突变体通过何种机制， 被蛋白酶体识别和降解;目的4阐明 高亲和力HERG通道阻滞剂纠正缺陷性 LQT 2突变体通道的蛋白质运输。我们将结合使用 生物化学、免疫组织化学和膜片钳技术研究野生型 在转染的组织培养细胞中表达的HERG和LQT 2突变通道， 在无细胞系统中。这些研究将加强我们的知识， 错误折叠和不当组装的LQT 2突变通道被识别， 保留和降解的ER质量控制系统，以及如何HERG通道 阻断剂修饰这些过程并拯救LQT 2突变通道。阐明 这些机制是发展的重要一步， 治疗先天性LQTS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.