PHOSPHORYLATION-DEPENDENT REGULATION OF CFTR CL-CHANNELS

囊性纤维化是由编码囊性纤维化的基因突变引起的。 纤维化跨膜传导调节因子（CFTR）。 CFTR是一种血浆 膜C1通道受磷酸化调节。 磷酸化 cAMP依赖性蛋白激酶在R结构域中的四个丝氨酸残基 激活通道。 磷酸化依赖性激活是 通过一种尚未鉴定的蛋白质 磷酸酶。 蛋白激酶C也激活CFTR C1通道，但 比PKA刺激更少的电流。 在这项研究中，我们将使用 电生理学和生物化学工具来了解 磷酸化和去磷酸化调节CFTR。 首先，学习如何 cAMP依赖性蛋白激酶激活CFTR C1通道，我们将使用 采用单通道膜片钳技术比较野生型 型和突变型CFTR C1通道。 我们将研究基因突变 单个通道上的四个可磷酸化丝氨酸残基中的每一个 打开概率和通道打开和关闭寿命。 这项工作 将帮助我们确定每个丝氨酸残基如何有助于CFTR C1- 渠道监管 第二，了解蛋白质磷酸化 激酶C激活CFTR C1通道，以及为什么它有影响 与PKA不同，我们将采用单通道膜片钳技术 和磷酸肽图谱，以确定哪些PKC磷酸化位点 负责激活通道。 最后，我们将确定 一种上皮磷酸酶，使CFTR C1- 渠道 我们将从上皮细胞中纯化酶，并研究 它的功能和规则。 这些研究的结果将提供 在分子水平上，我们对CFTR C1通道 是受控制的，因此，跨上皮C1分泌是如何调节的。

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.