Modulation and Regulation of ROMK Channels in Kidney

肾脏 ROMK 通道的调节和调节

• 批准号: 7486203
• 负责人: Wenhui Wang
• 金额: $ 26.74万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7486203
• 关键词: Arginine; Attenuated; Binding Sites; Cell membrane; Cyclic AMP-Dependent Protein Kinases; Data; Duct (organ) structure; Endocytosis; Goals; Intake; KCNJ1 gene; Kidney; Limb structure; Lysine; Mediating; Mono-S; Monoubiquitination; Mutation; Numbers; Oocytes; Phosphorylation; Phosphorylation Site; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Recycling; Regulation; Role; Surface; Testing; Thick; Tyrosine Phosphorylation; Ubiquitin; Ubiquitination; apical membrane; inward rectifier potassium channel; response; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The main goal of the present study is to explore the role of mono-ubiquitination of ROMK in the regulation of ROMK channel activity in response to stimulation of protein kinase A (PKA) and protein tyrosine kinase (PTK). ROMK is an inwardly-rectifying K channel and responsible for K recycling across the apical membrane of the thick ascending limb (TAL) and K secretion in the cortical collecting duct (CCD). The ROMK channel has three putative PKA-phosphorylation sites and one PTK-phosphorylation site. Stimulation of PKA-induced phosphorylation of ROMK channel augments the channel activity whereas increases in tyrosine-phosphorylation decrease the ROMK channel activity. Our preliminary data have shown that ROMK1 can be mono-ubiquitinated. Two lines of evidence indicate that lysine residue 22 (Lys22) is an ubiquiting binding site of ROMK1: 1) mutation of Lys22 to arginine increases the channel activity and surface expression of ROMK1; 2) ubiquitination of ROMK1 is absent in oocytes injected with R1K22R. Also, stimulation of PKA decreases the mono-ubiquitination of ROMK whereas low K intake, which has been shown to increase PTK activity, enhances the ubiquitination of ROMK. We will test the hypothesis that the mono-ubiquitination of ROMK1 is involved in the regulation of ROMK channel number in the cell membrane and that the ubiquitination process of ROMK1 is modulated by PKA and PTK. Specific Aim 1 To determine the role of mono-ubiquitination of ROMK in mediating the internalization and to examine whether Nedd4, a ubiquitin ligase E3, is involved in mediating mono-ubiquitination of ROMK. Specific Aim 2 To examine whether mono-ubiquitination of ROMK channels is regulated by PKA and PTK. Specific aim 3 To explore the role of CD63 in mediating ROMK1 tyrosine phosphorylation and mono-ubiquitination.

描述(申请人提供)：本研究的主要目的是探索ROMK的单一泛素化在蛋白激酶A(PKA)和蛋白酪氨酸激酶(PTK)刺激下调节ROMK通道活性中的作用。ROMK是一种内向整流性钾通道，负责钾在粗大升支(TAL)根尖膜上的循环和大脑皮层集合管(CCD)的钾分泌。ROMK通道有三个假定的PKA磷酸化位点和一个PTK磷酸化位点。刺激PKA诱导的ROMK通道的磷酸化增强了ROMK通道的活性，而增加酪氨酸磷酸化则降低了ROMK通道的活性。我们的初步数据表明ROMK1可以是单一泛素化的。有两条证据表明，赖氨酸残基22(Lys22)是ROMK1的泛素化结合位点：1)Lys22突变为精氨酸增加了ROMK1的通道活性和表面表达；2)注射R1K22R的卵母细胞不存在ROMK1的泛素化。此外，PKA的刺激降低了ROMK的单一泛素化，而低K摄入量则增加了PTK的活性，增强了ROMK的泛素化。我们将验证这样的假设，即ROMK1的单一泛素化参与了细胞膜上ROMK通道数量的调节，并且ROMK1的泛素化过程受到PKA和PTK的调控。 具体目的1确定ROMK的单一泛素化在介导ROMK内化中的作用，并检测泛素连接酶E3 Nedd4是否参与了ROMK的单一泛素化。 具体目的2检测ROMK通道的单一泛素化是否受PKA和PTK的调节。 特异性目的3探讨CD63在介导ROMK1酪氨酸磷酸化和单泛素化中的作用。