ALTERNATE C1 SECRETORY PATHWAYS IN CYSTIC FIBROSIS

描述：拟议研究的主要目标是了解 分子、免疫和生理水平的机制， 调节离子渗透通过导电Cl-通道存在于 分泌上皮细胞顶膜，并阐明其 与囊性纤维化跨膜传导的潜在相互作用 调节器（CFTR）。除CFTR外的上皮Cl-通道的研究 主要是因为缺乏重要的目标， 囊性纤维化（CF）的药物治疗。本实验室曾 成功地分离、纯化和重建了一种140 kDa的蛋白质 牛气管中的一种对DIDS敏感的阴离子通道。 该应用程序有两个具体目标：（1）直接测试 假设从牛气管纯化的天然140 kDa蛋白质 作为调节的钙激活的Cl-通道（CaCC）起作用。这些 研究人员将检查某些生化特性的， 蛋白质，包括糖基化的程度，它的能力， 被蛋白激酶A（PKA），蛋白激酶C（PKC）， 酪氨酸激酶（TK）和Ca 2 +/钙调蛋白依赖性蛋白激酶 （CaMK），以及这些磷酸化的功能后果 平面脂质双层中的反应。其他生物物理特性 天然和生物化学修饰的通道如离子选择性， 还将确定药理学抑制和动力学;（2） 为了鉴定和表征对应于所述基因的全长cDNA， 包含牛气管140 kDa Cl-通道的多肽， 为了证实这种蛋白质确实起到离子通道的作用， 并鉴定人cDNA同源物。调查人员将检查 由所述蛋白质编码的蛋白质的生物化学和功能表达， cDNA表达文库，并分析免疫纯化的蛋白质， 从生物合成的角度来看。这些研究将进一步促进我们的 生理学、生物化学和分子生物学知识 这些重要的氯离子转运途径的性质，并增加我们的 了解气道上皮细胞的液体分泌， 可以设计CF的替代治疗的潜在途径， 评估。

DESCRIPTION: The main goal of the proposed research is to understand at the molecular, immunological, and physiological level the mechanisms and regulation of ion permeation through conductive Cl- channels present in the apical membrane of secretory epithelia, and to elucidate their potential interactions with the cystic fibrosis transmembrane conductance regulator (CFTR). Studies of epithelial Cl- channels other than CFTR have been limited, largely because of the lack of important targets for pharmacological therapy in cystic fibrosis (CF). This laboratory has successfully isolated, purified, and reconstituted a 140 kDa protein from bovine trachea that functions as a DIDS-sensitive anion channel. The application has two specific aims: (1) to test directly the hypothesis that the native 140 kDa protein purified from bovine trachea functions as a regulated, calcium-activated Cl- channel (CaCC). These investigators will examine certain biochemical characteristics of the protein including the extent of glycosylation, its ability to be phosphorylated by protein kinase A (PKA), protein kinase C (PKC), tyrosine kinases (TK), and Ca2+/calmodulin-dependent protein kinases (CaMK), as well as the functional consequences of these phosphorylation reactions in planar lipid bilayers. Other biophysical properties of native and biochemically modified channels such as ion selectivity, pharmacological inhibition, and kinetics will also be determined; (2) to identify and characterize full-length cDNAs corresponding to the polypeptides comprising the 140 kDa Cl- channel of bovine trachea in order to verify that this protein indeed functions as an ion channel, and to identify a human cDNA homolog. The investigators will examine the biochemical and functional expression of the protein encoded by the cDNA expression libraries, and analyze immunopurified protein from the point of view from its biosynthesis. These studies will further our knowledge of the physiological, biochemical, and molecular biological properties of these important Cl- transport pathways and increase our understanding of fluid secretion across airway epithelia so that potential avenues of alternate therapy in CF can be devised and evaluated.