ALTERNATE C1 SECRETORY PATHWAYS IN CYSTIC FIBROSIS

描述：拟议研究的主要目标是了解 在分子、免疫和生理水平上的机制和 离子通过导电氯离子通道的渗透调节 分泌上皮细胞的顶膜，并阐明其 与囊性纤维化跨膜电导的潜在相互作用 调整器(CFTR)。非CFTR型上皮氯离子通道的研究 都是有限的，主要是因为缺乏重要的目标 囊性纤维化的药物治疗。这个实验室有 成功分离、纯化和重组140 kDa蛋白 来自牛的气管，起着DDS敏感的阴离子通道的作用。 该应用程序有两个具体目标：(1)直接测试 从牛气管中提纯的天然140 kDa蛋白的假设 作为一种受调节的、钙激活的氯离子通道(CACC)。这些 调查人员将检查细菌的某些生化特征 蛋白质包括糖基化的程度，它的能力 蛋白激酶A(PKA)、蛋白激酶C(PKC)、 酪氨酸激酶(TK)和钙/钙调素依赖性蛋白激酶 (CaMK)，以及这些磷酸化的功能后果 平面脂质双层中的反应。的其他生物物理性质 天然和生化修饰的通道，例如离子选择性， 药理抑制和动力学也将被确定；(2) 识别和鉴定与该基因对应的全长cDNA 牛气管140 kDa氯离子通道多肽的研究 为了验证这种蛋白质确实起到离子通道的作用， 并鉴定出一个人的同源基因。调查人员将检查 该基因编码蛋白的生化和功能表达 C DNA表达文库，并对免疫纯化蛋白进行分析 从其生物合成的角度来看。这些研究将进一步推动我们的 生理、生化和分子生物学知识 这些重要的氯离子运输途径的性质和增加我们的 了解跨呼吸道上皮细胞的液体分泌，以便 可以设计出治疗CF的替代疗法的潜在途径，并 已评估。

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.