MOLECULAR BIOLOGY OF CYSTIC FIBROSIS

我们将利用我们多学科CF研究小组的优势 研究cftr的加工、贩运和功能， 单个结构域，与其他离子通道的相互作用，以及其他 CL通道通路。项目I：研究替代中环线 频道。目的是确定人类ClC-2Cl-通道是否 可以改善CF中的Cl-缺陷。具体目标将：1.完成 克隆人类形式，2.表征ClC-2产生的氯电流， 3.确定clc-2的细胞定位；4.确定其作用。 CLC-2在呼吸道细胞中的表达。项目II：CF中的Na+和阳离子通道。 在CF患者中，Na+重吸收异常高。我们的目标是 为了评价阳离子通道与cftr之间的相互作用，2. 评估核苷酸门控阳离子通道是否对Na+有贡献 在CF中重吸收；3.克隆并鉴定药理作用 和肠道核苷酸门控阳离子的电生理特性 频道。项目三：非洲技术研究中心与人道协调委员会的互动。CFTR规范 外向整流氯离子通道(ORCC)。以下问题将 1.ORCC和CFTR之间的偶联机制是什么？ 2.CFTR的哪些结构域对调控至关重要 CFTR和ORCC之间的相互作用。3.哪些氨基酸定义了毛孔 Cftr.项目四：CFTR的功能分析。Cf中未知的密钥 是哪些氨基酸排列在CFTRCl-通道孔中。两个目标 将实现：1.确定对氯离子通道重要的氨基酸 活性和2.识别可能作为盐桥相互作用的残基 在CFTR中。项目V：在酵母中折叠和运输STE6和CFTR。 Cftr和酿酒酵母转运蛋白STE6是一个 三磷酸腺苷结合盒蛋白超家族。主要目标包括：1. STE6错折叠突变体的分离和鉴定 膜插入、折叠、质量控制、运输、 和ABC蛋白的降解，3.ste6-cftr嵌合体的制造 研究折叠和质量控制机械，4.高水平表达 5.探索STE6的分子内结构。项目 VI：原生cftr的产生和载体鉴定。其中一个 阻碍功能研究的问题是缺乏足够的数量 Cftr.目标是：1；共表达cftr和分子伴侣 在杆状病毒/昆虫系统中，2.使用快速繁殖的寄生虫 生物作为真核过表达系统，3.利用大肠杆菌 大量生产CFTR，4.将提纯的CFTR加入 脂质体囊泡和5.确定磷酸化的程度， 三磷酸腺苷的水解和反离子流是CFTR功能所必需的。那里 将有两个核心：分子生物学和管理学。

We will utilize the strengths of our multidisciplinary CF Research Group to study the processing, trafficking and function of CFTR, the role of individual domains, the interaction with other ion channels, and other Cl- channel pathways. Project I: Investigation of Alternate Cll Channels. The goal is to determine whether the human ClC-2 Cl- channel can ameliorate the Cl- defect in CF. Specific aims will: 1. complete the cloning of the human form, 2. characterize ClC-2 generated Cl- currents, 3. determine the cellular location of ClC-2, and 4. determine the role of ClC-2 in airway cells. Project II: Na+ and Cation Channels in CF. Na+ reabsorption is abnormally high in patients with CF. The goals are to 1. to evaluate the interaction between cation channels and CFTR, 2. to evaluate whether nucleotide-gated cation channels contribute to Na+ reabsorption in CF, and 3. to clone, then characterize the pharmacologic and electrophysiologic properties of intestinal nucleotide-gated cation channels. Project III: Interaction of CFTR with ORCC. CFTR regulates outwardly rectifying Cl- channels (ORCCs). The following questions will be addressed: 1. What is the mechanism of coupling between ORCC and CFTR? 2. What structural domains of CFTR are critical for the regulatory interaction between CFTR and ORCCs. 3. Which amino acids define the pore of CFTR. Project IV: Functional Analysis of CFTR. A key unknown in CF is which amino acids are lining the CFTR Cl- channel pore. Two goals will be achieved: 1. identify amino acids important to Cl channel activity and 2. to identify residues that may interact as salt bridges in CFTR. Project V: Folding and Trafficking of STE6 and CFTR in Yeast. CFTR and the Saccharomyces cerevisiae transporter STE6 are members of a superfamily of ATP binding cassette proteins. Major goals include: 1. the isolation of misfolding mutants of STE6, 2. identification of the mechanism of membrane insertion, folding, quality control, trafficking, and degradation of ABC proteins, 3. manufacture of STE6-CFTR chimeras to study folding and quality control machinery, 4. high level expression of CFTR, and 5. probing the intramolecular architecture of STE6. Project VI: Production and Vectorial Characterization of Native CFTR. One of the problems with hamper functional studies is a lack of sufficient amounts of CFTR. The goals are to: 1; coexpress CFTR and molecular chaperones in baculoviral/insect systems, 2. use rapidly proliferating parasitic organisms as eukaryotic overexpression systems, 3. utilize E. coli to produce large quantities of CFTR, 4. incorporate purified CFTR into liposomal vesicles and 5. determine the extent to which phosphorylation, ATP hydrolysis and counterion flow are required for CFTR function. There will be two cores: Molecular Biology and Administrative.