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C1膜通道。此外，一种新的基因 染色体上缺陷基因定位的探讨 7将利用ION中使用的筛选方法 交通研究。培养的细胞将贯穿整个过程 求婚。在项目1(Widdicombe)中，第二信使的级别、 正常人和慢性萎缩性胃炎的蛋白激酶及其靶蛋白 我们将对细胞进行比较。蛋白质磷酸化的变化将 与C_1分泌的变化相比较。在项目2(葡萄酒)中， 将使用各种不同的方法来研究对C1通道的调节 包括膜片钳在内的技术。重点将放在 确定在呼吸道中发现的相同的C1通道缺陷是否可以 在其他受影响的上皮细胞中显示，并且关于是否超过 一种通道类型受到影响。在项目3(Verkman)中，条例 将在重建后进行研究 脂质体或平面脂双层。根尖膜囊泡来自 呼吸道培养物和其他组织将被用作C1的来源 频道。不过，我们将尝试净化c1通道。 这不是成功重组的必要条件。在……里面 重建研究，C1通道被有效分离 从其他顶膜蛋白中分离出来，提供了一种直接的方法 测试其在CF中是否有缺陷。临床研究将是 在斯坦福大学和人类的临床/细胞获取中心进行 每所大学的细胞培养核心。临床/细胞 收购核心公司(刘易斯顿)将为 USCF文化核心(Finkbeiner)。我们将努力实现转型 并提高细胞的分化水平 电生理学研究揭示的培养。最终， 临床/细胞采集核心将应用所获得的信息 在基础科学项目中对CF患者进行治疗。

The research proposed here concentrates on the regulation of apical membrane C1 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 C1 secretion. In Project 2 (Wine), regulation of C1 channels will be studied using a variety of techniques including patch-clamping. Emphasis will be placed on determining if the same C1 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 C1 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 C1 channels. Attempts will be made to purify the C1 channel, though this is not necessary for successful reconstitution. In reconstitution studies, the C1 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.