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BIOCHEMISTRY OF AMILORIDE SENSITIVE NA+ CHANNELS IN CF

CF 中阿米洛利敏感 NA 通道的生物化学

基本信息

批准号：
2017338
负责人：
SUBRAHMANYESWARA U RAO
金额：
$ 9.5万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2002-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2017338
关键词：
MDCK cell Sf9 cell line amiloride chloride channels cystic fibrosis ion transport phosphorylation protein purification protein structure function respiratory epithelium sodium channel

项目摘要

Cystic fibrosis (CF) is a fatal disease of abnormal epithelial ion- transport, characterized principally by reduced Cl- and increased amiloride-sensitive Na+ conductances in pulmonary epithelia. Since the gene coding for the cystic fibrosis conductance regulator (CFTR) was identified, intense efforts have been directed toward understanding the malfunction of Cl- secretion. However, the abnormalities associated with Na+ transport are poorly understood. Recent identification of alpha, beta and gamma subunits that are presumed to constitute the functional epithelial Na+ channel (ENaC) makes possible the mechanistic studies of the abnormally raised and cAMP regulated Na+ transport associated with CF. In heterologous systems, ENaC activity is high and stimulated by cAMP, whereas, in the presence of CFTR, ENaC is down- regulated and cAMP effects reversed mimicking the in vivo observations of CF and normal airway epithelia, respectively. These observations form the basis for the long term goal of this grant application. Two heterologous expression systems, MDCK and Sf9 insect cells expressing ENaC and CFTR, will be utilized to undertake a detailed biochemical investigation of the structure and regulation of the ENaC. Utilizing these experimental systems, a research plan has been organized around 3 specific aims. 1. We shall isolate the functional ENaC and determine its composition and stoichiometry of the subunits. 2. We shall investigate the mechanism by which cAMP stimulates the ENaC. We shall identify the amino acid residues that are phosphorylated in the ENaC, and correlate the phosphorylation events to the function by site- directed mutagenesis. 3. We shall determine the mechanism by which CFTR regulates ENaC function. We shall investigate the hypothesis that CFTR regulates the ENaC by the mechanisms involving phosphorylation and dephosphorylation of amino acids in the ENaC subunits. We shall also test the hypothesis that physical interactions of CFTR with ENaC are responsible for down-regulation of ENaC function by immunological methods. It is anticipated that this research will aid further in understanding the ENaC-mediated Na+ absorption process across the reabsorbing epithelia and ameliorating the diseases associated with ENaC dysfunction.

囊性纤维化（CF）是一种上皮细胞异常的致死性疾病，运输，其特征主要是减少Cl-和增加肺上皮细胞中阿米洛利敏感的Na+电导。以来编码囊性纤维化传导调节因子（CFTR）的基因，确定，紧张的努力已被导向了解， Cl-分泌障碍。然而，与之相关的异常与Na+转运的关系知之甚少。最近的研究发现 α、β和γ亚基，它们被认为构成了功能性上皮钠离子通道（ENaC）使机制成为可能 Na ~+转运异常升高及cAMP调节的研究与CF有关。在异源系统中，ENaC活性高，在cAMP的刺激下，而在CFTR的存在下，ENaC下降- 调节和cAMP作用逆转模拟体内观察 CF和正常气道上皮细胞。这些观察结果这是本基金长期目标的基础。两异源表达系统，MDCK和Sf 9昆虫细胞表达 ENaC和CFTR将用于进行详细的生物化学研究ENaC的结构和调节。利用这些实验系统，一个研究计划已经组织围绕 3具体目标 1.我们将分离功能性ENaC并确定它的组成和亚基的化学计量。 2.我们将研究cAMP刺激ENaC的机制。我们将鉴定ENaC中磷酸化的氨基酸残基，并将磷酸化事件与位点的功能联系起来定向诱变 3.我们将确定CFTR 调节ENaC功能。我们将研究CFTR 通过磷酸化和磷酸化机制调节ENaC ENaC亚基中氨基酸的去磷酸化。我们亦会测试CFTR与ENaC的物理相互作用是负责通过免疫调节下调ENaC功能方法. 预计这项研究将有助于进一步了解ENaC介导的Na+吸收过程，重吸收上皮细胞和改善与ENaC相关的疾病功能障碍