Role of Molecular Chaperones in hERG Processing

分子伴侣在 hERG 处理中的作用

• 批准号: 6872166
• 负责人: ECKHARD K FICKER
• 金额: $ 33.75万
• 依托单位: METROHEALTH MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6872166
• 关键词: cardiac myocytes; developmental genetics; endoplasmic reticulum; gene mutation; immunoprecipitation; laboratory mouse; laboratory rabbit; long QT syndrome; mass spectrometry; molecular chaperones; potassium channel; proteasome; protein biosynthesis; protein degradation; protein folding; protein structure function; site directed mutagenesis; tissue /cell culture; ubiquitin; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): The overall goals are to identify and understand the function of molecular chaperones essential for productive folding, retention and degradation of hERG potassium channels during biosynthesis in the endoplasmic reticulum. The hERG gene encodes the rapid component of the cardiac delayed rectifier current IKr that is crucial for cardiac repolarization and critical to the normal duration and propagation of the cardiac action potential. Mutations in hERG produce functionally impaired or trafficking-deficient channels that reduce IKr current and are linked to hereditary long QT syndrome type2 in which delayed repolarization is associated with torsade de pointes and sudden cardiac death in young people. The specific aims of this study are to: (1) identify the molecular components of the multi-chaperone folding machinery associated with hERG wildtype channels during synthesis, assembly and maturation in the endoplasmic reticulum (ER), (2) probe remodeling of the multi-chaperone folding machinery associated with misprocessed LQT2 hERG mutations retained in the ER, (3) study the relationship of hERG-chaperone complexes with the ubiquitin/proteasome system and determine how triage decisions towards protein degradation are made, and (4) validate in native cardiomyocytes the physiological role for hERG chaperones and components of the ubiquitin/proteasome system identified in heterologous expression systems. An important outcome of this study will be the identification of novel molecular targets that can be exploited to restore trafficking of misfolded LQT2 mutants or increase the folding propensity of wildtype hERG channels to stabilize impaired cardiac action potentials. The research uses pulse-chase labeling, immunoprecipitation, autoradiography and immunoblotting to isolate and characterize the multi-chaperone machinery associated with hERG potassium channels. Mass spectrometry is used to identify novel protein components of the cellular chaperone machinery as well as of the proteasomal degradation machinery associated with hERG potassium channels. Patch-clamp electrophysiology, mutagenesis and adenoviral gene transfer of dominant-negative chaperone constructs are used to manipulate chaperone expression in heterologous expression systems as well as in native cardiomyocytes.

描述（由申请人提供）：总体目标是识别和理解在内质网生物合成过程中 hERG 钾通道的有效折叠、保留和降解所必需的分子伴侣的功能。 hERG 基因编码心脏延迟整流电流 IKr 的快速分量，这对于心脏复极至关重要，并且对于心脏动作电位的正常持续时间和传播至关重要。 hERG 突变会产生功能受损或运输缺陷的通道，从而降低 IKr 电流，并与遗传性长 QT 综合征 2 型相关，其中延迟复极与年轻人的尖端扭转型室性心动过速和心源性猝死有关。本研究的具体目的是：（1）在内质网（ER）合成、组装和成熟过程中鉴定与 hERG 野生型通道相关的多伴侣折叠机制的分子成分，（2）探针重构与 ER 中保留的错误处理的 LQT2 hERG 突变相关的多伴侣折叠机制，（3）研究 hERG 伴侣与泛素/蛋白酶体系统复合，并确定如何对蛋白质降解做出分类决策，以及 (4) 在天然心肌细胞中验证异源表达系统中鉴定的 hERG 伴侣和泛素/蛋白酶体系统组件的生理作用。这项研究的一个重要成果将是鉴定新的分子靶标，这些靶标可用于恢复错误折叠的 LQT2 突变体的运输或增加野生型 hERG 通道的折叠倾向，以稳定受损的心脏动作电位。该研究使用脉冲追踪标记、免疫沉淀、放射自显影和免疫印迹来分离和表征与 hERG 钾通道相关的多伴侣机制。质谱法用于鉴定细胞伴侣机制以及与 hERG 钾通道相关的蛋白酶体降解机制的新蛋白质成分。显性失活伴侣结构的膜片钳电生理学、诱变和腺病毒基因转移用于操纵异源表达系统以及天然心肌细胞中的伴侣表达。