CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) CHLORIDE CHANNEL

囊性纤维化是由于上皮细胞减少， 由于编码囊泡的基因突变导致的Cl-渗透性 纤维化跨膜电导调节因子（CFTR）Cl-通道。 CFTR通道，像ATP结合蛋白家族的所有其他成员一样， 盒（ABC）转运蛋白，包含两个核苷酸结合 结构域（NBD），但也包括一个独特的调控R结构域 含有10个以上的磷酸化共有位点， cAMP依赖性蛋白激酶（PKA）和蛋白激酶C（PKC）。 在 具有正常CFTR通道的细胞，受体介导的PKA活化 引起几个R-结构域丝氨酸的磷酸化，允许通道 通过涉及ATP水解的循环打开和关闭。 我们有 最近获得的强有力的证据表明ATP水解为细胞提供能量 构象变化，打开通道门， 通道磷酸化的程度是决定 大门就一直开着 我们的假设是磷酸化 特定的丝氨酸控制，独立地，这两个功能 NBD。 在具有两个NBD功能的强磷酸化通道中， 然后ATP在一个NBD处的水解打开通道， 第二个ATP可以在另一个NBD处结合，并且这样做可以稳定 开放构象 第二个ATP的水解， 稳定，促使通道关闭。 我们还假设 不同的细胞磷酸酶差异地去磷酸化 各种磷酸丝氨酸。 因此，在细胞中，激活或抑制 特定的磷酸酶可能有助于复杂的机制， 调节通道门控的 这个项目的目的是学习 哪些丝氨酸在哪个实验条件下被磷酸化， 目的是最终辨别出每一个人的确切角色 磷酸丝氨酸在协调个体通道功能中的作用 域. 方法是将生物化学信息与 通过精确测定单通道功能进行磷酸化 水平 传统上，磷酸化丝氨酸的鉴定 通过胰蛋白酶消化，然后用2-D磷酸肽 作图和直接测序或与适当的 （丝氨酸-丙氨酸）突变肽。 壮观的初步结果 使用质谱法获得，以鉴定 然而，磷酸肽表明，这将是我们的方法， 选择 该方法已经表明（a）Ser 768既是 最容易在限制ATP水平磷酸化的残基， 最容易被纯化的磷酸酶2A去磷酸化的残基，（B） Ser 737也容易被磷酸化， 磷酸化可能改变R结构域的构象， (c)丝氨酸670，以前没有显示是PKA的靶标， 在体外被PKA磷酸化。 这些早期结果表明， 明确的位点特异性突变靶点和详细的功能 分析. 我们目前正在进行非常详细的MS研究， 磷酸化和去磷酸化（具有许多不同的 磷酸酶），以便更全面地定义通道的成本 通过R域进行传导。 这项工作涉及综合利用一个 多种方法，包括MALDI-TOF-MS，MALDI-ITMS和 LC-ESI-ITMS/MS。现阶段为详细的平行功能性 和生化研究，在野生型和突变型CFTR通道中， 特定磷酸丝氨酸在单通道门控中的作用， 它们对选择性磷酸酶的敏感性。

The disease cystic fibrosis results from reduced epithelial Cl-permeability due to mutations in the gene encoding the cystic fibrosis transmembrane conductances regulator (CFTR) Cl- channel. CFTR channels, like all other members of the family of ATP-binding cassette (ABC) transporters, incorporates two nucleotide binding domains (NBDs) but also includes a unique regulatory R domain containing more than 10 consensus sites for phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). In cells with normal CFTR channels, receptor-mediated activation of PKA causes phosphorylation of several R-domain serines, permitting channel opening and closing via cycles involving ATP hydrolysis. We have recently obtained strong evidence that ATP hydrolysis energizes the conformational change that opens the channel gate, and that the degree of phosphorylation of a channel is one of the determinants of how long the gate stays open. Our working hypothesis is that phosphorylation of particular serines controls, independently, the function of the two NBDs. In a strongly phosphorylated channel with both NBDs functional, then hydrolysis of ATP at one NBD opens the channel, whereupon a second ATP can bind at the other NBD and in so doing can stabilize the open conformation. Hydrolysis of that second ATP then abolishes the stabilization, prompting channel closure. We have also hypothesized that distinct cellular phosphatases differentially dephosphorylate the various phosphoserines. Hence, in the cell, activation or inhibition of specific phosphatases could contribute to the complex mechanisms that regulate channel gating. The aim of this project is to learn which serines are phosphorylated under which experimental condition, with the goal of eventually discerning the exact role of each phosphoserine in orchestrating the function of the individual channel domains. The approach is to correlate biochemical information on phosphorylation with precise assays of function at the single channel level. Traditionally, identification of phosphorylated serines has been accomplished by tryptic digestion followed by 2-D phosphopeptide mapping and either direct sequencing or comparison with appropriate (serine-alanine) mutant peptides. The spectacular initial results obtained using mass spectrometry to identify phosphorylated serines in phosphopeptides, however indicate that this will be our method of choice. The method has already shown (a) that Ser 768 is both the residue most readily phosphorylated at limiting ATP levels and the residue most readily dephosphorylated by purified phosphatase 2A, (b) that Ser 737 is also readily phosphorylated and that its phosphorylation likely alters the conformation of the R domain, and (c) that Ser 670, not previously shown to be a target of PKA, is indeed phosphorylated by PKA in vitro. These early results suggest obvious targets for site-specific mutation and detailed functional analysis. We are currently carrying out very detailed MS studies of both phosphorylation and dephosphorylation (with a number of different phosphatases) in order to more ccompletely define costs of chanel conducatance via the R-domain. This work involves integrated use of a variety of proceudres including MALDI-TOF-MS, MALDI-ITMS and LC-ESI-ITMS/MS. The stage is now set for detailed parallel functional and biochemical studies, in both wild- type and mutant CFTR channels, of the roles of specific phosphoserines in single-channel gating and of their sensitivities to selective phosphatases.