Opening and Closing Mechanisms of CFTR Channels

描述（由申请人提供） CFTR（囊性纤维化跨膜传导调节因子）， CF中突变的基因，是形成Cl-离子通道的ABC蛋白。开业 和关闭是由ATP结合和CFTR水解的循环控制的 两个核苷酸结合结构域（NBD），其功能反过来由 CFTR中蛋白激酶A介导的多种丝氨酸磷酸化 监管领域。家长资助的目标是了解，在分子 详细介绍了NBD的结构和功能机制， 它们之间的关系，以及它们被调节的机制。为实现这一目标 父母格兰特利用电生理学，生物化学，分子， 生物学和生物物理学方法（包括结构建模和， 希望，最终晶体学）来表征WT的功能， 突变CFTR蛋白，在广泛的条件下。FIRCA的提案旨在 用于微观门控的额外详细测量和分析的资金 动力学主要由Csanady博士在国外研究中心进行 （匈牙利布达佩斯的塞梅尔维斯大学医学院）。该附加 拟议的工作将大大扩展和提高分析能力的 父母NIH赠款R 01 DK 51767，通过开发和测试适当的强大的 马尔可夫动力学计划，将解释所有数据的功能WT和 突变CFTR通道，无论是在亲本或外源位点获得（或实际上 任何调查人员在任何地方获得的所有可靠数据）。正在进行的关闭， P1和Csanady博士之间建立了富有成效的工作关系， 过去大约5年（后者在洛克菲勒攻读博士学位期间） 确保所提议的合作工作取得成功。美国集团将 提供分子生物学和生物化学支持：具体而言，我们将 向Csanady博士提供纯化PKA催化亚基、WT和突变体的cDNA CFTR，并提供整体的生化和电生理 这些和其他CFTR构建体的表征。在国外，博士。 Csanady将在卵母细胞中表达cDNA，进行CFTR的详细测量， 在被设计为 强调诊断能力，并使用他的 定制开发的软件，利用最大似然优化， 目的是得到最合适的马尔可夫方案，以充分描述， 定量地，控制CFTR打开和关闭的过程 通道孔这两个小组将密切互动，以最大限度地取得进展， 两个项目。

DESCRIPTION (provided by applicant) CFTR (cystic fibrosis transmembrane conductance regulator), the product of the gene mutated in CF, is an ABC protein that forms a Cl- ion channel. Its opening and closing are controlled by cycles of ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBDs) whose function, in turn, is regulated by protein-kinase-A-mediated phosphorylation of multiple serines in CFTR's regulatory domain. The goal of the parent grant is to understand, in molecular detail, the structure and mechanisms of function of the NBDs, the interactions between them, and the mechanisms by which they are regulated. Towards that goal the parent grant exploits electrophysiological, biochemical, molecular biological, and biophysical methods (including structural modeling and, hopefully, eventually crystallography) to characterize the function of WT and mutant CFTR protein, over a wide range of conditions. This FIRCA proposal seeks funds for additional detailed measurements and analysis of microscopic gating kinetics to be carried out primarily by Dr. Csanady at the foreign site (Semmelweis University Medical School, in Budapest, Hungary). This additional proposed work will greatly extend and enhance the analytical power of the parent NIH grant R0l DK51767, by developing and testing appropriate powerful Markov kinetic schemes that will explain all data on the function of WT and mutant CFTR channels, whether obtained at the parent or foreign site (or indeed all reliable data obtained by any investigator anywhere). The ongoing close, productive, working relationship developed between the P1 and Dr. Csanady over the past approximately 5 years (during the latter's PhD work at Rockefeller) ensures that the proposed collaborative work will succeed. The U.S. group will provide molecular biological and biochemical support: specifically, we will supply Dr. Csanady with purified PKA catalytic subunit, cDNAs of WT and mutant CFTRs, and provide overall biochemical and electrophysiological characterizations of those and other CFTR constructs. At the foreign site, Dr. Csanady will express the cDNAs in oocytes, make detailed measurements of CFTR channel gating kinetics in excised patches under conditions designed to emphasize diagnostic power, and analyze those kinetics using his custom-developed software that harnesses maximum likelihood optimizations, with the aim of deriving the most appropriate Markov schemes to fully describe, quantitatively, the processes that control opening and closing of the CFTR channel pore. The two groups will closely interact to maximize the progress of both projects.