Molecular Mechanisms of CFTR Regulation

CFTR调控的分子机制

• 批准号: 7035846
• 负责人: JOHN A.A. LADIAS
• 金额: $ 25.56万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035846
• 关键词: X ray crystallography; chloride channels; membrane activity; molecular biology; phosphorylation; protein protein interaction; protein purification; protein structure function; recombinant proteins; sodium hydrogen exchanger; syntaxin

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated chloride channel that determines the rate of electrolyte and fluid transport in the apical membrane of epithelial cells. Abnormal CFTR function is associated with the pathogenesis of cystic fibrosis and secretory diarrhea. Our long-term objective is to understand the molecular mechanisms underlying the regulation of CFTR at the atomic level and develop novel strategies for modulating the activity of this channel and treating the CFTR-associated diseases. The CFTR topology consists of two membrane-spanning domains and five cytoplasmic domains: an N-terminal domain (NTD), two nucleotide-binding domains, a regulatory domain (R) and a C-terminal domain (CTD). The CFTR activity is modulated through phosphorylation of the R domain, ATP hydrolysis by the NBDs, and interactions of its NTD and CTD domains with syntaxin 1A and NHERF proteins, respectively. However the regulatory mechanisms remain unknown primarily because the three-dimensional structure of the CFTR domains and the structural basis of their interaction with intracellular regulatory proteins remain elusive. This proposal addresses these questions and focuses on the structural analysis of cytoplasmic CFTR domains and their complexes with regulatory proteins, using molecular biology techniques and X-ray crystallography. The specific aims are: 1. To dissect the structural basis of CFTR channel gating mediated through the interaction of the CFTR CTD with the NHERF PDZ1 and PDZ2 domains. 2. To elucidate the molecular mechanisms underlying the regulation of CFTR channel activity through the interaction of the CFTR NTD with syntaxin 1A. 3. To determine the three-dimensional atomic structures of the CFTR NBD1 and NBD2 domains. These studies will provide the first high-resolution three-dimensional structures of four cytoplasmic CFTR domains and the structural basis of CFTR regulation by proteins syntaxin 1A and NHERF. This information is an essential step towards elucidating the basic molecular mechanisms that control the CFTR channel gating. Importantly, the atomic coordinates of these complexes could be used for structure-based rational design of drugs that would modify selectively the CFTR activity with clinical applications in the treatment of cystic fibrosis and secretory diarrhea.

描述（由申请人提供）：囊性纤维化跨膜电导调节剂（CFTR）是ATP调节的氯化物通道，它确定上皮细胞顶部膜中电解质和流体转运速率。异常CFTR功能与囊性纤维化和分泌性腹泻的发病机理有关。我们的长期目标是了解CFTR在原子水平上调节的分子机制，并制定调节该通道活性并治疗CFTR相关疾病的新型策略。 CFTR拓扑由两个跨膜结构域和五个细胞质结构域组成：一个N末端结构域（NTD），两个核苷酸结合域，一个调节域（R）和一个C末端结构域（CTD）。 CFTR活性通过R结构域，NBD的ATP水解以及其NTD和CTD结构域与语法1A和NHERF蛋白的相互作用进行调节。但是，调节机制仍然未知，主要是因为CFTR结构域的三维结构以及它们与细胞内调节蛋白相互作用的结构基础仍然难以捉摸。该建议解决了这些问题，并着重于使用分子生物学技术和X射线晶体学的细胞质CFTR结构域及其与调节蛋白的复合物的结构分析。具体目的是： 1。通过CFTR CTD与NHERF PDZ1和PDZ2域的相互作用介导的CFTR通道门控的结构基础。 2。阐明通过CFTR NTD与语法1a的相互作用调节CFTR通道活性的基础机制。 3。确定CFTR NBD1和NBD2域的三维原子结构。 这些研究将提供四个细胞质CFTR结构域的第一个高分辨率三维结构，以及蛋白质语法1A和NHERF的CFTR调节的结构基础。该信息是阐明控制CFTR通道门控的基本分子机制的重要步骤。 重要的是，这些复合物的原子坐标可用于基于结构的药物理性设计，这些设计将在治疗囊性纤维化和分泌性腹泻时有选择地修饰CFTR活性。