MOLECULAR PATHOPHYSIOLOGY OF CYSTIC FIBROSIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 囊性纤维化跨膜传导调节因子（CFTR）是一种蛋白激酶A（PKA）激活的ATP门控氯离子通道。上皮细胞顶膜中该通道的功能缺陷是囊性纤维化患者衰弱症状的原因。尽管CFTR的调节（R）结构域的PKA依赖性磷酸化对CFTR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。需要磷酸化多少丝氨酸残基才能激活CFTR？哪些残留物是必需的？这些磷酸化位点在功能上是退化的还是不同的？ 通过药理学试剂对CFTR调节的生物物理学研究已经得出结论，膜双层性质在CFTR功能中起关键作用。初步研究表明，胆固醇是细胞膜中的关键脂质成分，对CFTR功能及其对药理学试剂的反应有重大影响。胆固醇如何影响CFTR？它是否与CFTR蛋白结合？胆固醇对CFTR门控的影响是否继发于膜流动性的改变？细胞膜上富含维生素E的微区是否也参与其中？ 一个由生物化学家、生物化学家、生物工程师和分子生物学家组成的多学科小组已经成立，以解决这些重要问题。将使用各种技术，包括定点诱变，细胞附着，切除的内面向外和全细胞配置的膜片钳技术，快速光释放的笼状cAMP和膜流动性测量与新的分子转子。该提案旨在1）研究CFTR功能的磷酸化依赖性调节的分子基础，以及2）研究胆固醇调节CFTR的生物物理和生化机制。我们的研究将清楚地揭示CFTR是如何受磷酸化机制和脂质环境的调控。所获得的信息不仅有助于从根本上了解CFTR的功能，还有助于开发囊性纤维化患者的新疗法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.