MOLECULAR PATHOPHYSIOLOGY OF CYSTIC FIBROSIS

囊性纤维化（CF）是由囊性纤维化跨膜传导调节因子（CFTR）突变引起的，CFTR是一种camp依赖的氯离子通道。deltaF508突变约占CF突变的70%，90%的CF患者中至少存在一个拷贝。deltaF508突变导致内质网CFTR降解增加。突变蛋白的很大一部分到达质膜，但响应cAMP的突变通道的开放概率（Po）远低于野生型（wt） CFTR。我们已经证明异黄酮染料木素可以使deltaF508-CFTR的camp依赖性Po增加约20倍。这种cAMP依赖性激活的增加表明染料木素可能在CF治疗中具有治疗益处。染料木素作用的分子基础以及wt-和deltaF508-CFTR在cAMP激活方面的差异尚不清楚。对于wt-和deltaF508-CFTR， camp依赖性CFTR激活的哪一步不同？涉及到什么细胞机制？这种功能性缺陷的分子基础是什么？造成缺陷的结构变化是什么？规避这个缺陷的合理策略是什么？为了解决这些问题，我们将使用结合生化，电生理，药理学和分子生物学方法的方法组合。这些将包括定点诱变、细胞附着、由内到外切除和膜片钳技术的全细胞结构、全细胞电容测量、反义敲除技术和体内磷酸化CFTR的磷酸化肽定位。在细胞和分子水平上，CFTR调控和CF发病机制的清晰图像有望从这一多学科方法中出现。通过使用药物、纯化激酶和纯化磷酸酶，研究野生型和deltaF508-CFTR通过磷酸化/去磷酸化的调控。磷酸酶2C的过表达和反义敲除将用于研究该磷酸酶的生理和病理生理作用。结合磷酸化丝氨酸的磷酸化肽定位和位点定向诱变，我们将确定单个磷酸化位点的作用及其在CF中的病理生理意义。更清楚地了解deltaF508-CFTR功能缺陷的分子机制将有助于设计CF治疗干预的药物，如染料木素。

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel. The deltaF508 mutation accounts for approximately 70 percent of CF mutations and at least one copy is present in 90 percent of CF patients. The deltaF508 mutation causes increased degradation of CFTR in the endoplasmic reticulum. A significant portion of the mutant protein reaches to the plasma membrane, but the open probability (Po) of the mutant channel in response to cAMP is much lower than that of wild-type (wt) CFTR. We have demonstrated that the isoflavone genistein can increase the cAMP-dependent Po of deltaF508-CFTR by approximately 20-fold. This increase in cAMP-dependent activation suggests that genistein may be of therapeutic benefit in the treatment of CF. The molecular basis for the effects of genistein and the differences in cAMP activation of wt- and deltaF508-CFTR are poorly understood. Which step(s) of the cAMP-dependent CFTR activation differs for wt- and deltaF508-CFTR? What cellular machinery is involved? What is the molecular basis for this functional defect? What are the structural changes that account for the defect? What is a rational strategy to circumvent this defect? To address these questions, we will use a combination of approaches that incorporate biochemical, electrophysiological, pharmacological and molecular biological methodologies. These will include site-directed mutagenesis, cell-attached, excised inside-out and whole-cell configurations of the patch-clamp technique, whole-cell capacitance measurement, antisense knockout technology and phospho-peptide mapping of in vivo phosphorylated CFTR. A clear picture of CFTR regulation and CF pathogenesis at a cellular and molecular level is expected to emerge from this multidisciplinary approach. By using pharmacological agents, purified kinases and purified phosphatases, regulation of wild-type and deltaF508-CFTR via phosphorylation/dephosphorylation will be investigated. Overexpression and antisense knockout of phosphatase 2C will be used to study the physiological and pathophysiological role of this phosphatase. Combining phospho-peptide mapping and site-directed mutagenesis of phosphorylated serines, we will identify roles for individual phosphorylation sites and their pathophysiological significance in CF. A clearer understanding of the molecular mechanism for the functional defect of deltaF508-CFTR will aid in the design of pharmacological agents, such as genistein for therapeutic intervention in CF.