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通道活性调控中的作用。ROMK是一个向内整流的钾通道，负责钾在厚升肢顶端膜（TAL）的再循环和皮质集管（CCD）中的分泌。ROMK通道有三个pka磷酸化位点和一个ptk磷酸化位点。刺激pka诱导的ROMK通道磷酸化增强了通道活性，而酪氨酸磷酸化的增加则降低了ROMK通道活性。我们的初步数据表明ROMK1可以被单泛素化。两种证据表明，赖氨酸残基22 （Lys22）是ROMK1的普遍结合位点：1)赖氨酸对赖氨酸的突变增加了ROMK1的通道活性和表面表达；2)注射R1K22R的卵母细胞中ROMK1泛素化缺失。此外，PKA的刺激降低了ROMK的单泛素化，而低K摄入量（已被证明可以增加PTK活性）增强了ROMK的泛素化。我们将验证ROMK1的单泛素化参与细胞膜内ROMK通道数的调节，以及ROMK1的泛素化过程受PKA和PTK调节的假设。

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.