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。大约70%的CF突变由deltaF508突变引起，90%的CF患者中至少存在一个拷贝。DeltaF508突变导致内质网中CFTR降解增加。很大一部分突变蛋白到达质膜，但突变通道对cAMP的反应开放概率(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依赖的Po增加约20倍。这种cAMP依赖的激活增加表明染料木素可能在治疗CF方面有疗效。金雀异黄素作用的分子基础以及wt-和deltaF508-cftr在cAMP激活上的差异尚不清楚。依赖cAMP的cftr激活的哪个步骤(S)对于wt-和deltaF508-cftr有不同？这牵涉到什么细胞机制？这种功能缺陷的分子基础是什么？造成这一缺陷的结构变化是什么？绕过这一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。包括膜片钳技术的定点突变、细胞贴附、内向外切除和全细胞构型、全细胞电容测量、反义基因敲除技术和体内磷酸化CFTR的磷酸肽图。通过这种多学科的方法，有望在细胞和分子水平上清楚地了解CFTR的调控和CF的发病机制。通过使用药物、纯化的激酶和纯化的磷酸酶，我们将研究野生型和deltaF508-CFTR通过磷酸化/去磷酸化的调节。磷酸酶2C的过表达和反义敲除将被用来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多肽图谱和定点突变，我们将确定单个磷酸化位点在CF中的作用及其病理生理意义。更清楚地了解deltaF508-CFTR功能缺陷的分子机制将有助于设计用于治疗CF的药物，如金雀异黄素。

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.