CFTR--ION CONDUCTION AND GATING MECHANISMS

拟议研究的长期目标是调查 导致Cl电导激活的事件序列， 表达CFTR的细胞，并了解致病突变是如何 改变对激活条件的敏感性。 囊性纤维化是什么？ 最常见的致命的，遗传性疾病的白种人， 每2500个新生儿中就有1个受到影响，这种疾病是由突变引起的。 在编码膜蛋白的基因中， 跨膜传导调节因子（CFTR）。 野生型CFTR已被 与cAMP激活的Cl-选择性 在各种细胞类型中的电导，直到最近， 这种Cl传导在表达致病性的细胞中是不存在的， 突变CFTRs，特别是deltaF 508，最常见的突变相关 患有严重的囊性纤维化 然而，我们已经证明，在非洲爪蟾中， 卵母细胞表达deltaF 508和其他突变CFTR与 cAMP可激活的Cl电导，其表现出显著降低的 对激活刺激的敏感性（毛喉素+ IBMX）。 最 重要的是，敏感性的降低与 囊性纤维化的严重程度在患者携带相应 突变。 我们建议详细描述Cl的活化 表达野生型和突变CFTR的非洲爪蟾卵母细胞的电导。 一 导致激活的事件的定量描述 氯离子电导可以揭示突变发生的点 activation. 具体目标是：1.为了表征连续的 cAMP激活卵母细胞Cl通道的反应步骤 表达CFTR。2.为了表征相关的传导特性， 野生型CFTR在卵母细胞中的表达。3.使用特定 突变，以评估五个推定的功能意义， CFTR的结构域。 这些研究的结果可以提供一个 设计合理的药物治疗以改善 CF的症状，这似乎主要是由于缺乏足够的氯 分泌物 这项建议旨在与一个 Mitchell Drumm提交的第二个R 01，强调细胞特异性 CFTR的表达和治疗方式的鉴定， 不同的细胞类型

The long-range goal of the proposed research is to investigate the sequence of events which leads to the activation of Cl conductance in cells expressing CFTR and to understand how disease-causing mutations alter the sensitivity to activating conditions. Cystic Fibrosis is the most common lethal, recessively inherited disease among caucasians, affecting nearly 1 in 2500 newborns, and the disease is caused by mutations in the gene coding for a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Wild type CFTR has been associated with the expression of a cAMP activated, Cl-selective conductance in a variety of cell types, and until recently it was thought that this Cl conduction was absent in cells expressing disease-causing, mutant CFTRs, particularly deltaF508, the most common mutation associated with severe cystic fibrosis. We have shown, however, that in Xenopus oocytes expression of deltaF508 and other mutant CFTRs is associated with a cAMP activatable Cl conductance which exhibits a markedly reduced sensitivity to an activating stimulus (forskolin + IBMX). Most importantly, the reduction in sensitivity was highly correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. We propose to characterize in detail the activation of Cl conductance in Xenopus oocytes expressing wild type and mutant CFTRs. A quantitative description of the events leading to the activation of chloride conductance should reveal the points at which mutations alter activation. The specific aims are: 1. To characterize the sequential reaction steps involved in the activation of Cl channels by cAMP in oocytes expressing CFTR. 2. To characterize the conduction properties associated with the expression of wild type CFTR in oocytes. 3. To use specific mutations to evaluate the functional significance of the five putative structural domains of CFTR. The results of these studies could provide a mechanistic basis for the design of a rational drug therapy to ameliorate the symptoms of C.F., which appear to be largely due to insufficient Cl secretion. This proposal has been designed to interact closely with a second R01 submitted by Mitchell Drumm which emphasizes cell-specific expression of CFTR and the identification of therapeutic modalities in different cell types.