CFTR--ION CONDUCTION AND GATING MECHANISMS

拟议研究的长期目标是调查 导致氯电导被激活的一系列事件 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 在高加索人中最常见的致命性隐性遗传病， 每2500名新生儿中就有1人受到影响，这种疾病是由突变引起的 在编码一种膜蛋白的基因中，囊性纤维化 跨膜电导调节器(CFTR)。野生型CFTR已被 与cAMP激活、氯离子选择性表达相关 多种细胞类型的电导，直到最近，人们还认为 这种氯传导在表达致病基因的细胞中是缺失的， 突变CFRR，特别是DeltaF508，这是最常见的相关突变 患有严重的囊性纤维症。然而，我们已经证明，在非洲爪哇 卵母细胞表达deltaF508和其他突变的CFTRs与 CAMP可激活的氯电导显著降低 对激活性刺激(Forskolin+IBMX)的敏感性。多数 重要的是，敏感度的降低与 患者携带相应囊性纤维化的严重程度 突变。我们建议对氯的活化进行详细的表征 表达野生型和突变型cftr的非洲爪哇卵母细胞的电导。一个 对导致激活的事件的定量描述 氯离子电导应揭示突变改变的点 激活。具体目标是：1.刻画序列 CAMP激活卵母细胞氯离子通道的反应步骤 表达cftr。2.表征相关的传导性质 野生型CFTR在卵母细胞中的表达。3.使用特定的 突变以评估五种推定基因的功能意义 CFTR的结构域。这些研究的结果可以提供一个 合理设计改善药物治疗的机制基础 C.F.的症状，似乎主要是由于氯缺乏引起的 分泌物。此提案旨在与 Mitchell Drumm提交的第二个R01，强调细胞特异性 Cftr的表达及其治疗方案的确定 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.