Role of Molecular Chaperones in hERG Processing

描述（由申请人提供）：总体目标是识别和理解分子伴侣在内质网生物合成过程中对hERG钾通道的生产性折叠、保留和降解至关重要的功能。hERG基因编码心脏延迟整流电流IKr的快速成分，IKr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通道，减少IKr电流，并与遗传性长QT综合征2型有关，其中延迟复极与青年人的点扭转和心源性猝死有关。本研究的具体目的是：(1)确定内质网（ER）合成、组装和成熟过程中与hERG野生型通道相关的多伴侣折叠机制的分子组成；(2)探测内质网中保留的错误加工LQT2 hERG突变相关的多伴侣折叠机制的重塑；(3)研究hERG-伴侣复合物与泛素/蛋白酶体系统的关系，并确定如何做出蛋白质降解的分类决策。(4)在原生心肌细胞中验证hERG伴侣和在异源表达系统中鉴定的泛素/蛋白酶体系统组分的生理作用。这项研究的一个重要成果将是鉴定新的分子靶点，这些靶点可以用来恢复错误折叠的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的新蛋白质成分，以及与hERG钾通道相关的蛋白酶体降解机制。膜片钳电生理、诱变和腺病毒基因转移的显性阴性伴侣蛋白结构被用于操纵伴侣蛋白在异源表达系统和原生心肌细胞中的表达。

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.