Quality Control of K+ Channel Biogenesis

K 通道生物发生的质量控制

• 批准号: 6554304
• 负责人: Diane M Papazian
• 金额: $ 32.83万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 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/6554304
• 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

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）：这项研究的长期目标是阐明质量控制的机制，以确保仅在细胞表面表达正确折叠和组装的钾通道。 钾通道在急诊室中采用其本地结构。严格的质量控制系统可以识别出未能折叠或正确组装的通道蛋白，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到异常功能特性可能破坏细胞生理的位置。拟议的研究重点是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保留在ER中？其次，哪些途径处置了ER保留的钾通道蛋白？蛋白酶体降解和良好的形成的作用是什么，与其他ER滞留蛋白的处置有关？拟议的研究与通道病的病因有关，例如长期QT综合征2，其中通道蛋白保留在ER中，并可能受到与ER相关的降解。我们将完成以下特定目的：（1）确定细胞质结构域在振动振荡和KV1.3钾通道生物发生过程中的ER保留和释放中的作用； （2）确定哺乳动物细胞用来处置ER的钾通道蛋白的机制； （3）比较哺乳动物细胞系和心室心肌细胞中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，以研究蛋白质成熟，稳定性，蛋白酶抑制剂。实验方法将包括对野生型和突变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。拟议的研究将提高我们对离子通道细胞生物学基本方面的了解，并提高我们对通道病的病因的理解。