PHOSPHORYLATION-DEPENDENT REGULATION OF CFTR CL-CHANNELS

囊性纤维化是由编码囊性纤维症的基因突变引起的。 纤维化跨膜电导调节剂(CFTR)。CFTR是一种等离子体 膜C_1-通道受磷酸化调节。磷酸化 CAMP依赖的蛋白激酶在R区的四个丝氨酸残基 激活通道。依赖于磷酸化的激活是 通过一种尚未鉴定的蛋白质的作用而可逆 磷酸酶。蛋白激酶C也激活CFTRC1-通道，但 与PKA相比，刺激电流更少。在这项研究中，我们将使用 电生理和生化工具来了解如何 磷酸化和去磷酸化调节cftr。首先，要了解如何 CAMP依赖的蛋白激酶激活CFTRc1-通道，我们将使用 单通道膜片钳技术比较野生型心肌细胞的活动 Cftr C1通道的类型和突变体。我们将研究突变的影响 单通道上四个可磷酸化的丝氨酸残基中的每一个 打开概率和通道上的打开和关闭寿命。这部作品 将帮助我们确定每个丝氨酸残基对CFTRc1的贡献- 航道整治。第二，了解蛋白质的磷酸化是如何 激酶C激活CFTRc1-通道，以及为什么它有这样的作用 与PKA不同，我们将使用单通道膜片钳技术 并绘制磷酸肽图以确定哪些PKC磷酸化位点 负责通道激活。最后，我们将确定 去磷酸化和失活CFTRc1-的上皮磷酸酶 频道。我们将从上皮细胞中提纯这种酶，并研究 它的功能和调控。这些研究的结果将提供 美国在分子水平上对CFTRC1-通道的新知识 因此，跨上皮细胞的C1-分泌是如何被调节的。

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a plasma membrane C1-channel regulated by phosphorylation. Phosphorylation of four serine residues in the R domain by CAMP-dependent protein kinase activates the channels. Phosphorylation-dependent activation is reversible through the action of an as yet unidentified protein phosphatase. Protein kinase C also activates the CFTR C1- channel, but stimulates less current than does PKA. In this study, we will use electrophysiological and biochemical tools to understand how phosphorylation and dephosphorylation regulate CFTR. First, to learn how CAMP-dependent protein kinase activates the CFTR C1- channel, we will use the single-channel patch-clamp technique to compare the activity of wild- type and mutant CFTR C1- channels. We will study the effect of mutating each of the four phosphorylatable serine residues on the single channel open probability and on channel open and closed lifetimes. This work will help us determine how each serine residue contributes to CFTR C1- channel regulation. Second, to learn how phosphorylation by protein kinase C activates the CFTR C1-channel, and why it has an effect different from PKA, we will use th single channel patch-clamp technique and phosphopeptide mapping to determine which PKC phosphorylation sites are responsible for channel activation. Finally, we will identify the epithelial phosphatase that dephosphorylates and inactivates CFTR C1- channels. We will purify the enzyme from epithelial cells, and study its function and regulation. The results of these studies will provide us with new knowledge, at the molecular level, of how CFTR C1- channels are controlled, and hence, how transepithelial C1-secretion is regulated.