CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHLORIDE CHANNEL

囊性纤维化跨膜电导调节剂氯离子通道

• 批准号: 6279521
• 负责人: DAVID C GADSBY
• 金额: $ 2.52万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6279521
• 关键词: biomedical resource; human tissue; nervous system; spectrometry

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.

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 最近获得了有力的证据表明，ATP水解激发了 打开通道门的构象变化， 通道的磷酸化程度是一个决定因素， 星门会开多久 我们的假设是 特定丝氨酸的磷酸化独立地控制了 两个NBD的功能。 在一个强磷酸化通道中， 两个NBD功能，然后在一个NBD的ATP水解打开 通道，因此第二个ATP可以结合在另一个NBD上， Ding可以稳定开放构象。 第二种水解 然后ATP取消稳定，促使通道关闭。 我们 还假设不同的细胞磷酸酶 使各种磷酸丝氨酸差异性地去磷酸化。 所以在 特定磷酸酶的细胞、激活或抑制可 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被磷酸化 在这种实验条件下， 识别每个磷酸丝氨酸在协调 个别频道的功能。 采取方法是 将磷酸化的生化信息与精确的 单通道水平的功能测定。