Role of Molecular Chaperones in hERG Processing

分子伴侣在 hERG 处理中的作用

• 批准号: 7392385
• 负责人: ECKHARD K FICKER
• 金额: $ 32万
• 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 2009-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7392385
• 关键词: Action Potentials; Anabolism; Autoradiography; Biogenesis; Cardiac; Cardiac Myocytes; Cell membrane; Chaperone Gene; Complex; Defect; Dominant-Negative Mutation; Electrophysiology (science); Endoplasmic Reticulum; Ethers; Ethyl Ether; Gene Mutation; Gene Transfer; Genes; Goals; Heart; Human; Immunoblotting; Immunoprecipitation; Inherited; Ion Channel; Label; Link; Long QT Syndrome; Mass Spectrum Analysis; Molecular; Molecular Chaperones; Molecular Target; Mutagenesis; Mutation; Numbers; Outcome Study; Pathway interactions; Physiologic pulse; Physiological; Potassium Channel; Process; Processed Genes; Property; Proteins; Pulse taking; Research; Research Personnel; Research Proposals; Role; System; Torsades de Pointes; Triage; Ubiquitin; chaperone machinery; multicatalytic endopeptidase complex; mutant; novel; patch clamp; prevent; programs; protein degradation; protein function; protein misfolding; research study; sudden cardiac death; trafficking; ubiquitin-protein ligase; voltage

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（由申请方提供）：总体目标是鉴定和了解内质网生物合成过程中hERG钾通道高效折叠、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的快速成分，该成分对心脏复极化至关重要，对心脏动作电位的正常持续时间和传播至关重要。hERG突变产生功能受损或运输缺陷的通道，减少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相关。这项研究的具体目标是：（1）鉴定在内质网（ER）中合成、组装和成熟期间与hERG野生型通道相关的多分子伴侣折叠机制的分子组分，（2）探测与ER中保留的错误加工的LQT 2 hERG突变相关的多分子伴侣折叠机制的重构，（3）研究hERG-伴侣蛋白复合物与泛素/蛋白酶体系统的关系，并确定如何对蛋白质降解进行分类决策，和（4）在天然心肌细胞中验证hERG分子伴侣和泛素/泛素结合蛋白组分的生理作用。在异源表达系统中鉴定的蛋白酶体系统。本研究的一个重要结果将是鉴定新的分子靶点，可用于恢复错误折叠的LQT 2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钾通道相关的多分子伴侣机制。质谱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型蛋白质组分。膜片钳电生理学，诱变和腺病毒基因转移的显性负性分子伴侣构建体被用来操纵分子伴侣在异源表达系统以及在天然心肌细胞中的表达。

项目成果

Carvedilol targets human K2P 3.1 (TASK1) K+ leak channels.

卡维地洛针对人类 K2P 3.1 (TASK1) K 泄漏通道。

• DOI: 10.1111/j.1476-5381.2011.01319.x
• 发表时间: 2011
• 期刊: British journal of pharmacology
• 影响因子: 7.3
• 作者: Staudacher,K;Staudacher,I;Ficker,E;Seyler,C;Gierten,J;Kisselbach,J;Rahm,A-K;Trappe,K;Schweizer,PA;Becker,R;Katus,HA;Thomas,D
• 通讯作者: Thomas,D