REGULATED CLC CL CHANNELS AND CL SECRETION AND CF

这项建议的长期目标是为了了解 CIC CI通道家族的成员如何与CFTR一起， 其功能是提供跨气道上皮的Cl-转运。 这些 替代通道可以提供用于CI传输的其他路线， CFTR有缺陷。 在成年人中发现了两个CIC CI通道， 和胎儿人肺，CIC-2G（也称为CIC-2 α）和CIC-3。 CIC- 2G已从兔胃和人肺中克隆， 实验室，并显示PKA和CaMKII调节和调节， CIC-2G和CIC-3通道都可能参与 在调节人肺中的Cl-转运中。 这项建议是为了 确定生理、功能、结构和调节 人肺中CIC Cl-通道的特性。 具体目标 主要是：1）识别CI渠道形式，确定CI渠道层次， CIC-2G和CIC-3在人肺中的位置。 这一目标是建立在 在人肺中发现CIC-2G和CIC-3后，以及 定量RT-PCR结果表明存在这些 成人和胎儿肺中的通道。 定量RT-PCR、肽抗体和原位杂交将 用于定量CIC通道在人体内的分布 2）确定共识的功能意义 在人肺CIC-2G CI-通道中的磷酸化位点。 这一目标 是建立在发现额外的PKA和CaMKII位点的基础上的 在人CIC-2G中，人CIC-2G CI-通道功能通过 这些蛋白激酶，以及缺乏这些蛋白激酶的突变体中缺乏激活， 3）定义CIC-2G CI-通道的功能意义， 将转染的上皮细胞与未转染的上皮细胞进行比较和对比。 人气道上皮细胞内源性通道的特性 其含有CIC通道，使用膜片钳。 这包括影响 蛋白激酶（PKA和CaMK II）和低细胞质外pH值。轻度 使用酰胺化催化的通道活化的化学方法 通过水溶性聚酰亚胺将研究作为一种新的 开发治疗膀胱癌药物的途径 纤维化患者

The long term goal of this proposal is directed towards understanding how members of the CIC CI-channel family, in conjunction with CFTR, function to provide CI- transport across airway epithelia. These alternative channels may provide other routes for CI- transport when CFTR is defective. Two CIC CI- channels have been identified in adult and fetal human lung, CIC-2G (also named CIC-2 alpha) and CIC-3. CIC- 2G has been cloned from rabbit stomach and human lung by this laboratory, and show PKA and CaMKII regulation and regulation by extracytoplasmic pH. Both CIC-2G and CIC-3 channels may be involved in regulated CI- transport in the human lung. This proposal is to determine the physiological, functional, structural, and regulatory properties of CIC CI- channels in the human lung. The specific aims are to: 1) Identify the CI- channel forms, determine the levels and location of CIC-2G and CIC-3 in the human lung. This aim is built upon the finding of CIC-2G and CIC-3 in the human lung, and quantitative RT-PCR results which demonstrate the presence of these channels in the adult as well as in the fetal human lung. Quantitative RT-PCR, peptide antibodies and in situ hybridization will be used to quantitate the distribution of CIC channels in the human and rabbit lung; 2) Determine the functional significance of consensus phosphorylation sites in the human lung CIC-2G CI-channel. This aim is build upon the finding of additional consensus PKA and CaMKII sites in human CIC-2G, activation of human CIC-2G CI-channel function by these protein kinases, and lack of activation in mutants lacking these sites; 3) Define the functional significance of CIC-2G CI- channels in transfected epithelial cells will be compared and contrasted with the properties of endogenous channels from human airway epithelial cells which contain CIC channels using patch clamp. This includes effects of protein kinases (PKA and CaMKII) and low extracytoplasmic pH. Mild chemical procedures for channel activation using amidation catalysed by water soluble carbodiimides will be investigated as a novel approach to development of pharmaceuticals for treatment of cystic fibrosis patients.