REGULATION OF APICAL MEMBRANE CL CHANNELS IN AIRWAYS

本文的研究内容主要集中在根尖的调节上。 呼吸道中的膜氯通道。此外，还有一种新的遗传方法 7号染色体上缺陷基因的定位将利用 离子传输研究中使用的筛选方法。培养细胞 将在整个提案中使用。在项目1(Widdicombe)中，标高 第二信使、蛋白激酶及其靶向磷蛋白的表达 将对正常细胞和CF细胞进行比较。蛋白质磷酸化的变化 将其与氯分泌的变化进行比较。在项目2(葡萄酒)中， 氯离子通道的调节将使用各种技术进行研究 包括膜片钳技术。重点将放在确定是否 在呼吸道中发现的相同的氯通道缺陷可以在其他 受影响的上皮细胞，以及是否有一种以上的渠道类型受到影响。 在项目3(Verkman)中，将研究氯通道的调节 在重组为脂质体或平面脂双层之后。心尖 来自呼吸道培养物和其他组织的膜泡将被用作 氯离子通道的来源。将尝试净化氯离子通道， 尽管这不是成功重组的必要条件。在……里面 重建研究，氯通道有效地与其他通道分离 顶膜蛋白，提供了一种直接测试其是否 配置文件有缺陷。临床研究将在临床/细胞中进行 斯坦福大学的收购核心和人类细胞培养核心 上大学。临床/细胞采购核心(刘易斯顿)将提供 USCF培养核心的呼吸道组织(Finkbeiner)。尝试次数将是 使细胞转化，提高细胞分化水平 电生理研究显示的培养物。归根结底， 临床/细胞采集核心将应用在基本 科学计划用于治疗囊性纤维瘤患者。

The research proposed here concentrates on the regulation of apical membrane Cl channels in airways. In addition a novel genetic approach to the localization of the defective gene on chromosome 7 will take advantage of the screening methods used in the ion transport studies. Cultured cells will be used throughout this proposal. In Project 1 (Widdicombe), levels of second messengers, protein kinases and their target phosphoproteins in normal and CF cells will be compared. Changes in protein phosphorylation will be compared with changes in Cl secretion. In Project 2 (Wine), regulation of Cl channels will be studied using a variety of techniques including patch-clamping. Emphasis will be placed on determining if the same Cl channel defect found in airways can be demonstrated in other affected epithelia, and on whether more than one channel type is affected. In Project 3 (Verkman), regulation of the Cl channel will be studied following reconstitution into liposomes or planar lipid bilayers. Apical membrane vesicles from airway cultures and other tissues will be used as sources of Cl channels. Attempts will be made to purify the Cl channel, though this is not necessary for successful reconstitution. In reconstitution studies, the Cl channel is effectively separated from other apical membrane proteins, providing a direct means of testing whether it is defective in CF. Clinical research will be performed at a clinical/cell acquisition CORE in stanford and human cell culture CORE at each university. The clinical/cell acquisition CORE (Lewiston) will provide airway tissues for the USCF Culture CORE (Finkbeiner). Attempts will be made to transform the cells and to improve the level of differentiation of the cultures as revealed by electrophysiological studies. Ultimately, the clinical/cell acquisition CORE will apply information obtained in the basic science projects to the treatment of patients with CF.