MOLECULAR PATHOPHYSIOLOGY OF CYSTIC FIBROSIS

囊性纤维化的分子病理生理学

• 批准号: 7723127
• 负责人: Tzyh-Chang Hwang
• 金额: $ 0.05万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723127
• 关键词: Affect; Binding; Biochemical; Biomedical Engineering; Cell membrane; Cells; Chloride Channels; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Development; Environment; Epithelial Cells; Functional disorder; Funding; Grant; Institution; Lipids; Measurement; Membrane; Membrane Fluidity; Molecular; Patch-Clamp Techniques; Phosphorylation; Phosphorylation Site; Pilot Projects; Play; Property; Proteins; Reagent; Regulation; Research; Research Personnel; Resources; Role; Secondary to; Serine; Site-Directed Mutagenesis; Source; Symptoms; Techniques; United States National Institutes of Health; apical membrane; base; cystic fibrosis patients; novel; novel therapeutics; response

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 囊性纤维化跨膜电导调节剂（CFTR）是蛋白激酶A（PKA）激活的ATP门控氯化物通道。该通道在上皮细胞的顶膜中的功能有缺陷，导致囊性纤维化患者的症状使人衰弱。尽管CFTR的调节（R）结构域的PKA依赖性磷酸化对于CFTR功能至关重要，但R结构域磷酸化如何激活CFTR的分子机制仍不清楚。需要磷酸化多少丝氨酸残基激活CFTR？哪些残留物至关重要？这些磷酸化位点在功能上退化还是不同？ 药理学试剂对CFTR调节的生物物理研究导致结论是，膜双层特性在CFTR功能中起着至关重要的作用。试验研究表明，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。胆固醇如何影响CFTR？它与CFTR蛋白结合吗？胆固醇对膜流动性改变继发的CFTR门控的影响吗？细胞膜的富含Cholesolol的微域是否涉及？ 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了一个多学科团队，以解决这些重要问题。将使用多种技术，包括斑块钳技术的细胞连接，内外和全细胞构型，笼中cAMP的快速光泽和膜流动性测量新颖的分子转子的快速光泽。该建议的目的是1）研究CFTR功能磷酸化依赖性调节的分子基础，以及2）研究胆固醇CFTR调节的生物物理和生化机制。清楚地了解了CFTR如何受到磷酸化机制和脂质环境的调节。获得的信息不仅将有助于对CFTR的功能的基本了解，而且还有助于为囊性纤维化患者开发新的治疗疗法。