MOLECULAR BIOLOGY OF CYSTIC FIBROSIS

我们将利用我们的多学科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 氯离子通道。 项目一：替代性Cll调查 渠道 目的是确定人类ClC-2 Cl-通道 可改善CF中Cl-缺陷。 具体目标将：1。完成 克隆人的形式，2。表征ClC-2产生的Cl-电流， 3.确定ClC-2的细胞位置，以及4.确定角色 气道细胞中ClC-2的含量。 项目II：CF中的Na+和阳离子通道。 CF患者的Na+重吸收异常高。这些目标是 到1.为了评估阳离子通道和CFTR之间的相互作用，2. 评估核苷酸门控阳离子通道是否有助于Na+ CF中的重吸收，以及3.来克隆，然后描述 肠核苷酸门控阳离子的电生理特性 渠道 项目三：CFTR与ORCC的互动。 CFTR调节 外向整流氯离子通道（ORCCs）。 以下问题将 被处理：1. ORCC和CFTR之间的耦合机制是什么？ 2. CFTR的哪些结构域对于调节 CFTR和ORCC之间的相互作用。3.哪些氨基酸定义了毛孔 的CFTR。 项目四：CFTR的功能分析。 CF中的一个关键未知数 是哪些氨基酸排列在CFTR的氯离子通道孔里。 两球 将实现：1.鉴定对Cl通道重要氨基酸 活动，2。鉴定可能作为盐桥相互作用的残基 在CFTR。 项目V：STE 6和CFTR在酵母中的折叠和运输。 CFTR和酿酒酵母转运蛋白STE 6是 ATP结合盒蛋白超家族。 主要目标包括：1. 错误折叠突变体STE 6的分离，2.识别 膜插入、折叠、质量控制、运输的机制， 和ABC蛋白的降解，3. STE 6-CFTR嵌合体的制备， 学习折页及质量控制机械，4.的高效表达 CFTR和5。探索STE 6的分子内结构。 项目 VI：天然CFTR的产生和矢量表征。 之一 阻碍功能研究的问题是缺乏足够的量 的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.利用快速繁殖的寄生虫 生物作为真核过表达系统，3.利用E.杆菌以 生产大量的CFTR，4.将纯化的CFTR掺入 脂质体囊泡和5.确定磷酸化的程度， CFTR功能需要ATP水解和逆流。 那里 将有两个核心：分子生物学和行政。

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.