Opening and Closing Mechanisms of CFTR Channels

描述(由申请人提供) CFTR(囊性纤维化跨膜电导调节剂)，是 在Cf中突变的基因，是一种形成氯离子通道的ABC蛋白。它的开业 和关闭是由cftr的ATP结合和水解循环控制的。 两个核苷酸结合域(NBD)，其功能依次受 蛋白激酶A介导的CFTRs中多种丝氨酸的磷酸化 监管领域。父母资助的目标是在分子水平上理解 详细介绍了NBDS的结构和功能机制，以及相互作用 它们之间的关系，以及它们受到监管的机制。朝着这个目标前进 家长赠款利用电生理、生化、分子 生物学和生物物理方法(包括结构建模和 希望，最终，结晶学)来表征WT和 突变的cftr蛋白，在广泛的条件下。FIRCA的这项提案寻求 用于微观浇注的额外详细测量和分析的资金 动力学主要由Csanady博士在异地进行 (匈牙利布达佩斯的塞梅尔韦斯大学医学院)。这是额外的 拟议的工作将极大地扩展和增强 母公司NIH授予R0l DK51767，通过开发和测试相应的强大功能 马尔可夫动力学方案，将解释所有关于WT和 突变的cftr通道，无论是在父站点还是在外部站点(或者实际上 任何调查人员在任何地方获得的所有可靠数据)。正在进行的收盘， P1和Csanady博士之间发展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) 确保拟议的协作工作取得成功。这家美国集团将 提供分子生物学和生化支持：具体地说，我们将 为Csanady博士提供纯化的PKA催化亚基、WT的cDNA和突变体 CFTRs，并提供全面的生化和电生理 这些和其他CFTR结构的特征。在国外的网站上，Dr。 Csanady将在卵母细胞中表达cDNA，并对cftr进行详细测量 在设计为以下条件的切除斑块中的通道门控动力学 强调诊断能力，并使用HIS分析这些动力学 定制开发的软件，利用最大似然优化， 目的是得出最合适的马尔可夫方案来完全描述， 定量地说，控制CFTR的打开和关闭的过程 海峡毛孔。这两个小组将密切互动，以最大限度地促进 两个项目都是。

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.