Quality Control of K+ Channel Biogenesis

K 通道生物发生的质量控制

基本信息

批准号：
6942780
负责人：
Diane M Papazian
金额：
$ 29.7万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-02 至 2006-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6942780
关键词：
cell line confocal scanning microscopy electrophysiology endoplasmic reticulum fluorescence microscopy membrane proteins potassium channel protein biosynthesis protein degradation protein folding protein structure protein transport

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the mechanisms of quality control that ensure that only properly folded and assembled potassium channels are expressed on the cell surface. Potassium channels adopt their native structures in the ER. Channel proteins that fail to fold or assemble properly are recognized by a stringent quality control system and retained in the ER. This system prevents transport of misfolded or incompletely assembled proteins to locations where aberrant functional properties could disrupt cellular physiology. The proposed research focuses on two aspects of potassium channel quality control. First, how are structurally immature, misfolded, or unassembled potassium channel proteins retained in the ER? Second, what pathways dispose of ER-retained potassium channel proteins? What are the roles of proteasomal degradation and aggresome formation, which have been implicated in the disposal of other ER-retained proteins? The proposed research is relevant to the etiology of channelopathies, such as Long QT Syndrome Type 2, in which channel proteins are retained in the ER and may be subjected to ER-associated degradation. We will accomplish the following specific aims: (1) to determine the role of cytoplasmic domains in ER retention and release during biogenesis of Shaker and Kv1.3 potassium channels; (2) to identify mechanisms used by mammalian cells to dispose of ER-retained potassium channel proteins; and (3) to compare the quality control of HERG channel biogenesis in a mammalian cell line and in cardiac ventricular myocytes. Channel proteins will be expressed in HEK293T cells or cultured ventricular myocytes for studies of protein maturation, stability, protease inhibitors. Experimental approaches will include expression and biochemical analysis of wild type and mutant channel proteins, confocal microscopy, cell surface labeling, and electrophysiology. The proposed research will advance our knowledge of basic aspects of ion channel cell biology and improve our understanding of the etiology of channelopathies.

描述（由申请人提供）：本研究的长期目标是阐明质量控制机制，确保只有正确折叠和组装的钾通道才能在细胞表面表达。钾通道采用内质网中的天然结构。无法正确折叠或组装的通道蛋白会被严格的质量控制系统识别并保留在 ER 中。该系统可防止错误折叠或不完全组装的蛋白质运输到异常功能特性可能破坏细胞生理学的位置。本研究重点关注钾通道质量控制的两个方面。首先，结构不成熟、错误折叠或未组装的钾通道蛋白如何保留在内质网中？其次，什么途径可以处理 ER 保留的钾通道蛋白？蛋白酶体降解和聚集体形成在其他 ER 保留蛋白的处理中起什么作用？拟议的研究与通道病的病因学相关，例如 2 型长 QT 综合征，其中通道蛋白保留在 ER 中，并可能遭受 ER 相关的降解。我们将实现以下具体目标：（1）确定Shaker和Kv1.3钾通道生物发生过程中细胞质结构域在ER保留和释放中的作用； (2) 确定哺乳动物细胞处理内质网保留的钾通道蛋白的机制； (3) 比较哺乳动物细胞系和心室肌细胞中 HERG 通道生物发生的质量控制。通道蛋白将在 HEK293T 细胞或培养的心室肌细胞中表达，用于研究蛋白成熟、稳定性、蛋白酶抑制剂。实验方法将包括野生型和突变型通道蛋白的表达和生化分析、共聚焦显微镜、细胞表面标记和电生理学。拟议的研究将增进我们对离子通道细胞生物学基本方面的了解，并提高我们对离子通道病病因学的理解。