CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHLORIDE CHANNEL

囊性纤维化是由上皮细胞减少引起的 编码囊状细胞的基因突变导致的CL通透性 纤维化通道是膜电导调节器(CFTR)氯离子通道。 Cftr通道，像ATP结合家族的所有其他成员一样 盒(ABC)转运体，结合两个核苷酸结合 域(NBD)，但也包括唯一的监管R域 包含10多个共同的位点，通过 CAMP依赖的蛋白激酶(PKA)和蛋白激酶C(PKC)。在……里面 具有正常CFTR通道的细胞，受体介导的PKA激活 导致几个R结构域丝氨酸的磷酸化，允许通道 通过涉及ATP水解的循环来打开和关闭。我们有 最近获得了强有力的证据表明，ATP水解能 打开通道门的令人愉快的变化，以及 通道的磷酸化程度是决定因素之一 大门能开多长时间。我们的工作假设是 特定丝氨酸的磷酸化独立地控制着 两个NBD的功能。在强烈磷酸化的通道中 两个NBD都是功能性的，那么一个NBD上的ATP水解会打开 通道，于是第二个ATP可以结合在另一个NBD上，并在 丁二醛可以稳定开放构象。第二步的水解度 然后，ATP取消了稳定，促使通道关闭。我们 还假设不同的细胞磷酸酶 对不同的磷酸丝氨酸进行差异化脱磷。因此，在 细胞，激活或抑制特定的磷酸酶可以 有助于调节通道门控的复杂机制。 这个项目的目的是了解哪些丝氨酸是磷酸化的。 在什么样的实验条件下，最终的目标是 辨别每一种磷酸丝氨酸在协调 各个通道域的功能。方法是 将磷酸化的生化信息与精确的 单通道级别的功能分析。

The disease cystic fibrosis results from reduced epithelial Cl-permeability due to mutations in the gene encoding the cystic fibrosis traiismembrane 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 conforinational 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 ding 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.