ALTERNATE CHLORIDE ION SECRETORY PATHWAYS IN CYSTIC FIBROSIS

囊性纤维化中的替代氯离子分泌途径

• 批准号: 6354720
• 负责人: DALE J BENOS
• 金额: $ 15.34万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6354720
• 关键词: Xenopus; Xenopus oocyte; chloride channels; chloride ion; cystic fibrosis; gastrointestinal absorption /transport; gene expression; gene targeting; genetic library; ion transport; laboratory mouse; laboratory rabbit; laboratory rat; membrane reconstitution /synthesis; molecular cloning; protein purification; protein reconstitution; protein structure function; transfection

The main goal of the proposed research is to understand the mechanisms and regulation of ion permeation through C1- channels other than the cystic fibrosis transmembrane conductance regulatory (CFTR) present in secretory epithelia. These alternative C1- channels may be useful and important targets for pharmacological therapy in cystic fibrosis (CF). Our laboratory has successfully isolated and cloned a protein from bovine trachea that behaves as a Ca2+ -sensitive C1- channel (CaCC), and has semi-purified and reconstituted an outwardly-rectified C1- channel (ORCC). This application has four specific aims: (1) to test the hypothesis that the translated bovine tracheal cDNA forms an anion channel of identical characteristics to the native protein, that the 38 kDa subunit of the native tracheal CaCC protein is the result of post- translational processing of the cloned 100 kDa CaCC cDNA product, and to determine the biochemical properties of both native and cloned CaCCs. The functional properties of the proteins will also be characterized following reconstitution into planar lipid bilayers or transfection into eukaryotic cells; (2) to identify a full-length cDNA corresponding to the human CaCC homolog and to characterize the translated protein. The molecular structure and function of the human homolog of the bovine CaCC will be determined by screening of appropriate human epithelial cDNA libraries; (3) to purify a protein that behaves as an ORCC from bovine tracheal apical membrane vesicles and to identify and characterize the full-length cDNA that encodes this protein. Candidate proteins will be used to raise polyclonal antibodies that will be used to screen a bovine tracheal cDNA expression library. The ultimate goal is to isolate a full-length cDNA that encodes an ORCC and to characterize the translated protein with the aim of understanding is potential interaction with CFTR and/or other ion channels; (4) to determine if heterologous intestinal specific expression of the CaCC can overcome the lethal intestinal obstruction found in the CF knockout mouse model. We will test the hypothesis that tissue specific expression of the bovine CaCC in the intestine will prevent the lethal consequences of intestinal obstruction by ameliorating the adverse effects of impaired chloride secretion in the intestine. These studies will further our knowledge of the physiological, biochemical, and molecular properties of these important C1- transport pathways and increase our understanding of fluid secretion across airway and intestinal epithelial so that potential avenues of alternate therapy in CF can be devised and evaluated.

这项研究的主要目的是了解 以及调节通过除C1通道以外的C1通道的离子渗透 囊性纤维化跨膜传导调节（CFTR）存在于 分泌上皮 这些替代的C1通道可能是有用的， 囊性纤维化（CF）药物治疗的重要靶点。 我们的实验室已经成功地分离和克隆了一种蛋白质， 牛气管作为Ca 2+敏感的C1通道（CaCC）， 并具有半纯化和重组的向外精馏的C1- 通道（ORCC）。 本申请有四个具体目的：（1）测试 翻译的牛气管cDNA形成阴离子的假设 与天然蛋白质具有相同特征的通道，38 天然气管CaCC蛋白的kDa亚基是后 克隆的100 kDa CaCC cDNA产物的翻译加工，和 以确定天然和克隆的CaCC两者的生物化学性质。 蛋白质的功能特性也将被表征 在重构成平面脂质双层或转染入 真核细胞;（2）鉴定对应于所述真核细胞的全长cDNA。 人CaCC同源物并表征翻译的蛋白质。 的 人源牛源性大肠杆菌同源物的分子结构和功能 CaCC将通过筛选适当的人上皮细胞来确定。 cDNA文库;（3）从cDNA文库中纯化表现为ORCC的蛋白质， 牛气管顶膜囊泡，并确定和 鉴定编码该蛋白的全长cDNA。 候选 蛋白质将用于产生多克隆抗体， 筛选牛气管cDNA表达文库。 最终目标是 分离编码ORCC的全长cDNA，并表征 以理解为目的的翻译蛋白是潜在的 与CFTR和/或其他离子通道的相互作用;（4）确定是否 CaCC的异源肠特异性表达可以克服 在CF敲除小鼠模型中发现的致死性肠梗阻。 我们 将检验牛的组织特异性表达 肠道中的CaCC将防止肠道疾病的致命后果。 通过改善受损的氯化物的不良反应来改善阻塞 肠内分泌物。 这些研究将进一步加深我们对 这些蛋白质的生理、生物化学和分子特性 重要的C1-转运途径，并增加我们对 液体分泌穿过气道和肠上皮， 可以设计和评估CF的替代疗法的途径。