CFTR: Probing the Gating Related Movements in the Pore

CFTR：探讨毛孔中的门控相关运动

• 批准号: 6904979
• 负责人: XUEHONG LIU
• 金额: $ 11.19万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6904979
• 关键词: Xenopus; active transport; adenosine triphosphate; anions; biological transport; chloride channels; conformation; cysteine; membrane structure; molecular probes; protein structure; protein structure function; thiols; voltage /patch clamp

DESCRIPTION (provided by applicant): My long-term career goal is to carry out research that is focused on the molecular basis of disease. My immediate goal is to advance my career by starting to build an independent research program under the guidance of two distinctive and well-established mentors. Dr. Dawson will provide supervision in all aspects of the career development plan and Dr. Larsson will contribute in designing and interpreting experiments on state-dependence of cysteine accessibility. The long-range goal of the proposed research is to determine if there are structural components in the transmembrane domains (TMDs) of CFTR channel that are involved in the conformational changes associated with channel gating. It is well established that channel gating is controlled by the intracellular domains, but results from several studies have suggested that the pore domain is an important part of the gating mechanism. Little is known, however, about how the binding and hydrolysis of ATP in the nucleotide binding domains are translated into either the movement or a conformational change in TMDs that opens or closes the channel pore. The proposed research will focus on three specific aims: 1) To identify residues exhibiting state-dependent accessibility to Au(CN)2-. 2) To investigate the effect of Au(CN)2- on the single-channel function in constructs exhibiting state-dependent accessibility to Au(CN)2-. 3) To investigate the effects of other thiol-directed reagents on the function of constructs that show state-dependent accessibility to Au(CN)2-. The working hypothesis that will guide the proposed research is that CFTR shares the fundamental functional features of the ATP Binding Cassette (ABC) family in which conformational changes in the TMDs occur during the transport process. The research strategy is to assay the effects of thiol-directed reagents on engineered cysteines in the TMD's of CFTR using inside-out or outside-out patches that will allow the access to both the intracellular and the extracellular solution.

描述（由申请人提供）：我的长期职业目标是开展专注于疾病分子基础的研究。我的近期目标是在两位独特且知名的导师的指导下开始建立一个独立的研究项目，以推进我的职业生涯。道森博士将在职业发展计划的各个方面提供监督，拉尔森博士将有助于设计和解释半胱氨酸可及性的状态依赖性实验。本研究的长期目标是确定 CFTR 通道的跨膜域 (TMD) 中是否存在参与通道门控相关构象变化的结构成分。众所周知，通道门控是由细胞内结构域控制的，但多项研究的结果表明，孔结构域是门控机制的重要组成部分。然而，人们对核苷酸结合域中 ATP 的结合和水解如何转化为 TMD 的运动或构象变化（从而打开或关闭通道孔）知之甚少。拟议的研究将重点关注三个具体目标：1）识别对 Au(CN)2- 表现出状态依赖性可及性的残基。 2) 研究 Au(CN)2- 对具有状态依赖性的 Au(CN)2- 可及性的构建体中单通道功能的影响。 3) 研究其他硫醇导向试剂对具有状态依赖性的 Au(CN)2- 可达性的构建体功能的影响。指导拟议研究的工作假设是，CFTR 具有 ATP 结合盒 (ABC) 家族的基本功能特征，其中 TMD 的构象变化在运输过程中发生。研究策略是使用内向外或外向外的贴片来分析硫醇导向试剂对 CFTR TMD 中工程化半胱氨酸的影响，这些贴片将允许进入细胞内和细胞外溶液。