Role of Molecular Chaperones in hERG Processing

分子伴侣在 hERG 处理中的作用

• 批准号: 7210746
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7210746
• 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(由申请人提供)：总体目标是确定和了解分子伴侣在内质网生物合成过程中对HERG钾通道的高效折叠、保留和降解所必需的功能。HERG基因编码心脏延迟整流电流Ikr的快速成分，它对心脏复极至关重要，对心脏动作电位的正常持续时间和传播至关重要。HERG基因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型有关，在2型遗传性长QT综合征中，延迟复极与年轻人尖端扭转性心动过速和心脏性猝死有关。本研究的具体目的是：(1)确定内质网(ER)中与HERG野生型通道合成、组装和成熟相关的多伴侣折叠机制的分子组成，(2)探测与错误处理的LQT2 HERG突变相关的多伴侣折叠机制的重塑，(3)研究Herg-伴侣复合体与泛素/蛋白酶体系统的关系，并确定如何对蛋白质降解做出分流决定，以及(4)在天然心肌细胞中验证在异源表达系统中确定的Herg伴侣和泛素/蛋白酶体系统的组成部分的生理作用。这项研究的一个重要结果将是识别新的分子靶点，这些靶点可以用来恢复错误折叠的LQT2突变体的运输，或者增加野生型HERG通道的折叠倾向来稳定受损的心脏动作电位。本研究使用脉冲追逐标记、免疫沉淀、放射自显影和免疫印迹等方法分离和鉴定了与HERG钾通道相关的多分子伴侣机制。质谱学被用来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的蛋白质成分。膜片钳电生理、显性-负性伴侣基因的突变和腺病毒基因转移被用来操纵伴侣蛋白在异源表达系统中的表达以及在天然心肌细胞中的表达。