REGULATED CLC CL CHANNELS AND CL SECRETION AND CF

这项提议的长期目标是为了理解 CIC CI渠道家族的成员如何与CFTR合作， 通过呼吸道上皮细胞提供CI转运的功能。这些 当出现以下情况时，替代信道可以提供用于CI传输的其他路线 CFTR有缺陷。在成人中发现了两个CIC CI-通道 人胎肺、CIC-2G(又称CIC-2α)和CIC-3。中投公司- 用此方法从兔胃和人肺中克隆了2G 实验室，并展示了PKA和CaMKII的调节和调节 胞质外pH。可能同时涉及CIC-2G和CIC-3通道 在人类肺中受调控的CI运输。这项建议是为了 确定生理、功能、结构和调节 人肺CIC-CI通道的特性具体目标 目的是：1)确定CI渠道形式，确定级别和 CIC-2G和CIC-3在人肺中的定位这个目标是建立起来的 在人肺中发现CIC-2G和CIC-3，以及 定量RT-PCR结果表明这些基因的存在 在成人和胎儿肺中的通道。 定量RT-PCR、多肽抗体和原位杂交将 用于定量测定CIC通道在人体内的分布 和兔肺；2)确定共识的功能意义 人肺CIC-2G CI通道的磷酸化位点。这一目标 是建立在发现更多共识的PKA和CaMKII位点的基础上 在人CIC-2G中，通过以下途径激活人CIC-2G CI通道功能 这些蛋白激酶，在缺乏这些的突变体中缺乏激活 站点；3)定义CIC-2G CI通道在 将转染腺病毒的上皮细胞与 人呼吸道上皮细胞内源性通道的特性 其中包含使用膜片钳的CIC通道。这包括效果 蛋白激酶(PKA和CaMKII)活性降低，胞外pH降低。温和的 催化酰胺化激活通道的化学方法 水溶性碳二亚胺将作为一种新型的研究对象 治疗囊性疾病的药物开发探讨 纤维化症患者。

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.