MECHANISMS, STRUCTURE, AND REGULATION OF CFTR'S NBFS

了解囊性纤维化的作用和调节机制 (Cf)跨膜电导调节器(CFTR)通道，我们将使用 电生理学和蛋白质生物化学(和质谱学) 在分子水平上研究它的功能，分子生物学和 用于操纵和分析其结构的结晶学，以便进行关联 通过这些功能测量。我们的目标是检查准确的 特定的激酶和磷酸酶在调控中的作用机制 (R)野生型突变体cftr的结构域，以调节其功能 核苷酸结合折叠(NBF)，进而影响构象 改变了控制离子流过通道孔的方式。我们将在此基础上 我们最近的发现是CFTR上不同的磷酸化位点， 易受不同的磷酸酶攻击，独立调节 两种NBF结合和水解ATP的能力。一个特别不稳定的人 磷酸化位点似乎控制着通道的时间长度 例如，保持开放，以便识别和药理上 靶向。，调节该部位的特定磷酸酶应该 允许由于以下原因而导致离子流动不足的病态细胞的“拯救” 突变的CFTR通道的表达；这包括那些失败的突变 为了达到足够数量的细胞表面，那些具有 减小的单通道电导，以及那些在 足够大的时间段。有两个具体目标。第一 回答了以下问题：NBF如何发挥作用，以及 两种NBF相互作用的机理是什么？工作假说是 这两种NBF是相似的，因为它们都能分解ATP，但 在功能和机制上不同，一个人在核苷酸上变得“活跃” 水解，另一种只需要核苷酸结合，可能反映 记录了初级序列的差异，因此是三维的 结构。第二个问题是：磷酸化是如何 的R结构域(以及在哪个或哪些站点)控制NBF1的功能 允许开通频道吗？一个额外的磷酸盐是如何 不稳定的站点？如果NBF1处的ATP水解导致通道打开， 但NbF2处的ATP水解会促使通道关闭，然后机制 根据承载有 Cf相关突变将是模棱两可的，除非打开和关闭 监控各个通道的速率。这就是我们要衡量的， 野生型和突变型通道。

To learn the mechanisms of function and regulation of the Cystic Fibrosis (CF) Transmembrane conductance Regulator (CFTR) channel, we will use electrophysiology and protein biochemistry (and mass spectrometry) to examine its function at the molecular level, and molecular biology and crystallography to manipulate and analyze its structure, for correlation with these functional measurements. The goal is to examine the precise mechanisms by which specific kinases and phosphatases act on the regulatory (R) domain of wild-type an mutant CFTR to regulate the function of its nucleotide binding folds (NBFs) which, in turn, effect the conformational changes that control ion flow through the channel pore. We will build on our recent findings that different phosphorylation sites on CFTR, susceptible to attack by distinct phosphatases, independently regulate the ability of the two NBFs to bind and hydrolyze ATP. A particularly labile phosphorylation site appears to control the length of time the channel stays open, for example, so that identifying, and pharmacologically targeting., the specific phosphatase that regulates that site ought to permit the "rescue" of diseased cells with inadequate ion flow due to expression of mutant CFTR channels; this includes those mutants that fail to reach the cell surface in sufficient numbers, those that have a diminished single-channel conductance, and those that are not open for a large enough fraction of time. There are two specific aims. The first addresses the questions: How do the NBFs function, and what are the mechanisms of interactions between the two NBFs? The working hypothesis is that the two NBFs are similar, in that they both hydrolyze ATP, but t hey differ in function and mechanism one becoming "active" upon nucleotide hydrolysis, the other requiring only nucleotide binding likely reflecting documented differences in primary sequence and, hence, three-dimensional structure. The second addresses the questions; How does phosphorylation of the R domain (and at which site or sites) control the function of NBF1 to permit channel opening? How does phosphorylaiton of an additional labile site (or sites)? If ATP hydrolysis at NBF1 causes channel opening, but ATP hydrolysis at NBF2 prompts channel closing, then the mechanism underlying any alteration of the open probability of a channel bearing a CF-associated mutation will be ambiguous unless the opening and closing rates of individual channels are monitored. That is what we will measure, for wild-type and mutant channels.