DETERMINANTS OF CARDIAC REPOLARIZATION

快速激活的延迟整流钾电流(IKR)和被认为编码它的人类乙醚相关基因(HERG)在心脏复极中起关键作用，HERG突变导致先天性长QT综合征(LQT-2)。HERG、IKR和LQT-2将在转基因的HEK293细胞和天然兔心肌细胞中利用分子和电生理学技术进行研究。具体目的1是研究HERG野生型和LQT-2突变型离子通道的细胞处理机制。我们以前已经发现了HERG蛋白加工机制中的正常步骤，以及一些LQT-2突变体的异常加工。我们将通过检验三个假设来扩展这些观察：a)一些LQT-2突变体的正常功能失败涉及多个加工步骤中的缺陷，b)水貂或水貂相关亚基的共表达改变了HERG野生型和LQT-2蛋白的运输，c)野生型HERG蛋白与水貂LQT突变(LQT-5)的共表达改变了HERG电流的表达。我们将确定发生这种情况的步骤。具体目标2是研究改变HERG蛋白生产和降解的细胞过程。与野生型Herg蛋白相比，我们将检验LQT-2突变蛋白迅速降解的假设，这对新描述的形成功能通道的LQT-2突变通道特别重要。这种周转增加，如果得到证实，将是LQT-2表型表达的一种新机制。我们还将检验N-连接的糖基化是表达的HERG蛋白稳定性的决定因素的假设。我们将测试温度作为LQT-2突变通道运输到表膜的成功和效率的决定因素的影响，并确定可能改变HERG蛋白运输的其他细胞过程。目的3研究野生型HERG与LQT-2突变型通道亚基的共组装机制。我们将检验这样一个假设，即蛋白质运输异常改变了显性负效应的表达。我们将测试水貂、水貂相关和水貂突变(LQT-5)通道在修改这方面的作用(如果有的话)。这项研究将增加我们对HERG离子通道加工和功能的分子机制的了解，更广泛地说，将对所有离子通道都有意义。更具体地说，它将与人类疾病LQT-2的机制特别相关。阐明这些区域的机制对于开发理解正常和异常心律失常发生的新策略以及抗心律失常治疗的新策略具有重要意义。

The rapidly activating delayed rectifier K- current (IKr), and the human ether-a-go-related gene (HERG) thought to encode it, play a key role in cardiac repolarization, and mutations in HERG cause congenital long QT syndrome (LQT-2). HERG, IKr and LQT-2 will be studied using molecular and electrophysiological techniques in transfected HEK293 cells and native rabbit myocytes. Specific aim 1 is to study cellular mechanisms of ion channel processing of HERG wild type and LQT-2 mutant channels. We have previously identified normal steps in the processing mechanism for HERG protein as well as abnormal processing for some LQT-2 mutants. We will extend these observations by testing three hypotheses: a) the failure of some LQT-2 mutants to function normally involves defects in multiple processing steps, b) co-expression of the minK or minK-related subunits modifies HERG wild type and LQT-2 protein trafficking, c) co-expression of wild type HERG protein with minK LQT mutations (LQT-5) alters the expression of HERG current. We will identify the steps where this occurs. Specific aim 2 is to study cell processes that modify HERG protein production and degradation. We will test the hypothesis that LQT-2 mutant proteins are degraded rapidly, compared with wild type HERG protein, which is of particular importance for newly described LQT-2 mutant channels that form functional channels. Such an increased turnover, if demonstrated, would be a novel mechanism for the expression of the LQT-2 phenotype. We will also test the hypothesis that N- linked glycosylation is a determinant of the stability of expressed HERG protein. We will test the effects of temperature as a determinant of the success and efficiency of trafficking of LQT-2 mutant channels to the surface membrane, and we will identify additional cell processes that might modify HERG protein trafficking. Specific aim 3 is to study the mechanisms of co- assembly of wild type HERG with LQT-2 mutant channel subunits. We will test the hypothesis that protein trafficking abnormalities alter expression of the dominant negative effect. We will test what role, if any, minK, minK-related, and minK mutant (LQT-5) channels have in modifying this. This research will increase our knowledge of molecular mechanisms of ion channel processing and function of HERG and more generally will have implications for all ion channels. More specifically it will have particular relevance to mechanisms of the human disease LQT-2. Elucidating mechanisms in these areas in important in developing new strategies for understanding normal and abnormal arrhythmogenesis and for new strategies for anti-arrhythmic therapies.