Modulation and Regulation of ROMK Channels in Kidney

描述（由申请人提供）：本研究的主要目的是探索ROMK的单泛素化在响应蛋白激酶A（PKA）和蛋白酪氨酸激酶（PTK）刺激的ROMK通道活性调节中的作用。ROMK是一种内向整流钾离子通道，负责钾离子穿过粗升支（TAL）顶膜的再循环和皮质集合管（CCD）中的钾离子分泌。ROMK通道具有三个推定的PKA磷酸化位点和一个PTK磷酸化位点。PKA诱导的ROMK通道磷酸化的刺激增强了通道活性，而酪氨酸磷酸化的增加降低了ROMK通道活性。我们的初步数据表明，ROMK 1可以被单泛素化。两条证据表明，赖氨酸残基22（Lys 22）是ROMK 1的泛素化结合位点：1）Lys 22突变为精氨酸增加了ROMK 1的通道活性和表面表达; 2）在注射R1 K22 R的卵母细胞中，ROMK 1不存在泛素化。此外，PKA的刺激降低了ROMK的单泛素化，而低K摄入，这已被证明是增加PTK活性，增强了ROMK的泛素化。我们将测试这一假设，即ROMK 1的单泛素化参与调节细胞膜中ROMK通道的数量，以及ROMK 1的泛素化过程是由PKA和PTK调节。 具体目的1确定ROMK的单泛素化在介导内化中的作用，并检测泛素连接酶E3 Nedd 4是否参与介导ROMK的单泛素化。 具体目的2研究ROMK通道的单泛素化是否受PKA和PTK的调节。 具体目的3探讨CD 63在ROMK 1酪氨酸磷酸化和单泛素化中的作用。

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.