ALTERNATE CHLORIDE ION SECRETORY PATHWAYS IN CYSTIC FIBROSIS

拟议的研究的主要目标是了解这些机制。 和调节离子通过除 囊性纤维化跨膜电导调节(CFTR)存在于 分泌上皮细胞。这些替代的c1通道可能是有用的 囊性纤维化药物治疗的重要靶点。 我们实验室成功地分离和克隆了一种蛋白质 牛的气管表现为一个钙敏感的C1通道(CACC)， 并经过半提纯和重组，得到一种向外精馏的C1- 频道(ORCC)。这个应用程序有四个具体目标：(1)测试 翻译的牛气管cdna形成阴离子的假说 与天然蛋白质特性相同的通道，即38 天然的气管CACC蛋白的KDA亚基是后 克隆的100 kDa CACC cDNA产物的翻译加工，以及 测定原生和克隆CaCCs的生化性质。 还将对这些蛋白质的功能特性进行表征 重组为平面脂质双分子层或转染入 真核细胞；(2)鉴定与该基因相对应的全长基因 人CACC同源物，并对翻译的蛋白进行鉴定。这个 牛的人类同源基因的分子结构和功能 CACC将通过筛查适当的人上皮细胞来确定 C DNA文库；(3)从 牛气管顶膜囊泡的鉴定和鉴定 鉴定编码该蛋白质的全长cDNA.侯选人 蛋白质将被用来产生多克隆抗体，这些抗体将用于 筛选牛气管表达文库。最终目标是 分离编码ORCC的全长cDNA并鉴定 以理解为目的的翻译蛋白质是潜在的 与CFTR和/或其他离子通道的相互作用；(4)确定 异源肠特异性表达CACC可克服 在CF基因敲除小鼠模型中发现致死性肠梗阻。我们 将检验这样一种假设，即牛的组织特异性表达 肠道中的CACC将预防肠道的致命后果 改善氯化物损伤的不良反应所致的梗阻 肠道的分泌物。这些研究将进一步加深我们对 这些物质的生理、生化和分子特性 重要的C1-运输途径和增加我们对 通过呼吸道和肠道上皮细胞的液体分泌 可以设计和评估治疗CF的替代治疗方法。

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.