Molecular Pathophysiology of Cystic Fibrosis

描述(由申请人提供)：囊性纤维化跨膜电导调节器(CFTR)是一种蛋白激酶A(PKA)激活的、ATP门控的氯离子通道。上皮细胞顶膜上这一通道功能的缺陷是囊性纤维化患者衰弱症状的原因。尽管依赖于PKA的CFTR调节(R)结构域的磷酸化对CFTR的功能至关重要，但R结构域的磷酸化如何激活CFTR的分子机制仍不清楚。有多少丝氨酸残基需要被磷酸化才能激活cftr？哪些残留物是必需的？这些磷酸化位点是功能退化的还是截然不同的？ 通过对药物对CFTR调控的生物物理研究，我们得出结论：膜的双层性质在CFTR功能中起着关键作用。初步研究表明，胆固醇是细胞膜中的关键脂质成分，对CFTR功能及其对药理试剂的反应有重大影响。胆固醇是如何影响CFTR的？它能与CFTR蛋白结合吗？胆固醇对CFTR门控的影响是否次于膜流动性的改变？细胞膜上富含胆红素的微区是否参与其中？ 已经组建了一个由生物化学家、生物物理学家、生物工程师和分子生物学家组成的多学科团队来解决这些重要问题。将使用多种技术，包括定点突变、细胞附着、内向外切除和全细胞配置的膜片钳技术，笼式cAMP的快速光释放和使用新型分子旋转器测量膜流动性。本研究的目的是：1)研究依赖于磷酸化的CFTR功能调节的分子基础；2)研究胆固醇对CFTR功能调节的生物物理和生化机制。我们的研究将清楚地了解CFTR是如何受到磷酸化机制和脂质环境的调节的。获得的信息不仅有助于从根本上了解CFTR是如何发挥作用的，而且还有助于开发治疗囊性纤维化患者的新疗法。

DESCRIPTION (provided by applicant): Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a protein kinase A (PKA)-activated, ATP-gated chloride channel. Defective function of this channel in the apical membrane of epithelial cells is responsible for the debilitating symptoms in patients with cystic fibrosis. Although PKA-dependent phosphorylation of the regulatory (R) domain of CFTR is critical for CFTR function, the molecular mechanism of how phosphorylation of the R domain activates CFTR remains unclear. How many serine residues need to be phosphorylated to activate CFTR? Which residues are essential? Are those phosphorylation sites functionally degenerate or distinct? Biophysical studies of CFTR modulation by pharmacological reagents have led to the conclusion that membrane bilayer properties play a critical role in CFTR function. Pilot studies show that cholesterol, a key lipid component in cell membranes, has a major impact on CFTR function and its response to pharmacological reagents. How does cholesterol affect CFTR? Does it bind to the CFTR protein? Is the effect of cholesterol on CFTR gating secondary to an alteration of membrane fluidity? Is the choleserol-rich microdomain of the cell membrane involved? A multi-disciplinary team with biochemist, biophysicist, bioengineer and molecular biologist has been assembled to tackle these important questions. A variety of techniques will be used including site-directed mutagenesis, cell-attached, excised inside-out and whole-cell configurations of the patch-clamp technique, rapid photorelease of caged cAMP and membrane fluidity measurements with novel molecular rotors. The proposal is aimed to 1) study the molecular basis for phosphorylation-dependent regulation of CFTR function, and 2) investigate the biophysical and biochemical mechanisms for CFTR modulation by cholesterol. A clear picture of how CFTR is regulated by phosphorylation machinery and lipid environment will emerge from our studies. The information obtained will not only facilitate a fundamental understanding of how CFTR functions, but also aid in the development of novel therapeutics for patients with cystic fibrosis.