Kir5.1 regulates Kir4.1 ubiquitination by Nedd4-2 in DCT

项目摘要/摘要 内向整流性K通道4.1(Kir4.1)由Kcnj10编码，在提供 基侧钾电导与远曲小管负膜电位的关系 (DCT)。Kir4.1在DCT中的关键作用被证明是功能缺失突变的报告 肾脏中的Kcnj10会引起肾小管病变，这种疾病让人想起吉特尔曼综合征，包括 低镁血症、低钾血症和代谢性碱中毒。肾功能丧失突变的肾脏表型 在芝麻/EAST综合征中观察到的KIR4.1在肾脏特异的Kir4.1基因敲除中被概括(Ks-Kir4.1 Ko)小鼠模型。我们已经证明Ks-Kir4.1 KO小鼠有严重的低钾血症和 低氯代谢性碱中毒，提示DCT膜转运有缺陷。KIR4.1相互作用 Kcnj16编码的Kir5.1在DCT的基底外侧膜上形成一个40pS的K通道。而当 KIR4.1负责在DCT中提供基侧K电导，Kir5.1可能起调节作用 异四聚体的亚基。Kir5.1包含一个基于苏氨酸的Nedd4 E3连接酶结合基序 它的C末端(TPVT)，已被证明是磷酸酶CDC25c中Nedd4的结合部位。这个 Kir5.1可能通过与Nedd4-2结合来调节DCT中Kir4.1的活性 我们的初步实验表明，Kir5.1和Nedd4-2的缺失会刺激Kir4.1的活性，这是 已知能刺激NCC活动。NCC不仅在调节DCT对Na的吸收中起作用，而且在DCT中也起着重要作用 调节肾脏钾排泄。因此，本申请的目标是检验Kir5.1是 Nedd4-2 E3连接酶的结合伙伴，它泛素化Kir4.1并去极化DCT中的膜 Kir5.1和Nedd4-2 E3连接酶在高钠高钾抑制效应中的关键作用 摄取基底侧的Kir4.1和根尖的NCC。该提案有三个具体目标：在具体目标1中， 我们将测试Kir5.1(Kcnj16)是否是Nedd4-2 E3连接酶的结合伙伴，以及Nedd4-2是否与 Kir5.1泛素化Kir4.1，去极化DCT中的膜。在具体目标2中，我们将测试 Kir5.1和Nedd4-2 E3连接酶参与高钠摄入抑制作用的假说 在DCT中的Kir4.1和NCC上。在特定的目标3中，我们将检验Kir5.1和Nedd4-2 E3连接酶的假设 在介导高K摄入量对DCT中Kir4.1和NCC的抑制作用中起重要作用。自.以来 了解Kir5.1在DCT中的作用，Kir4.1在调节WNK-Spak-NCC中起关键作用 而Nedd4-2对Kir4.1的调控将为钾和钠转运的调控提供一个完整的观点 在远端小管中。

Project Summary/Abstract Inwardly-rectifying K channel 4.1 (Kir4.1) is encoded by Kcnj10 and plays a dominant role in providing basolateral K conductance and in determining the negative membrane potential in the distal convoluted tubule (DCT). The critical role of Kir4.1 in the DCT is demonstrated by the report that loss-of-function mutations of Kcnj10 in the kidney cause tubulopathy, a disease which is reminiscent to Gitelman's syndrome including hypomagnesemia, hypokalemia and metabolic alkalosis. The renal phenotype of loss-function mutations of Kir4.1 observed in SeSAME/EAST syndrome is recapitulated in the kidney-specific Kir4.1 knockout (Ks-Kir4.1 KO) mouse model. We have demonstrated that Ks-Kir4.1 KO mice have severe hypokalemia and hypochloremic metabolic alkalosis, suggesting a defective membrane transport in the DCT. Kir4.1 interacts with Kir5.1 encoded by Kcnj16 to form a 40 pS K channel in the basolateral membrane of the DCT. While Kir4.1 is responsible for providing basolateral K conductance in the DCT, Kir5.1 may serve as a regulatory subunit for the heterotetramer. Kir5.1 contains a phosphor-threonine-based Nedd4 E3 ligase binding motif at its c-terminus (TPVT), which has been shown to be a binding site of Nedd4 in phosphatase CDC25c. The possibility that Kir5.1 may regulate Kir4.1 activity in the DCT through binding to Nedd4-2 is also suggested by our preliminary experiments showing that depletion of Kir5.1 and Nedd4-2 stimulates Kir4.1 activity which is known to stimulate NCC activity. NCC not only plays a role in mediating Na absorption in the DCT but also in regulating renal K excretion. Thus, the goal of the present application is to test hypothesis that Kir5.1 is the binding partner for Nedd4-2 E3 ligase which ubiquitinizes Kir4.1 and depolarizes membrane in the DCT and that Kir5.1 and Nedd4-2 E3 ligase play a key role in mediating the inhibitory effects of high Na and high K intake on the basolateral Kir4.1 and the apical NCC. The proposal has three specific aims: In specific aim 1, we will test whether Kir5.1(Kcnj16) is a binding partner of Nedd4-2 E3 ligase and that Nedd4-2 binding to Kir5.1 ubiquitinizes Kir4.1 and depolarizes the membrane in the DCT. In specific aim 2, we will test the hypothesis that Kir5.1 and Nedd4-2 E3 ligase are essential for mediating the inhibitory effect of high Na intake on Kir4.1 and NCC in the DCT. In specific aim 3, we will test the hypothesis that Kir5.1 and Nedd4-2 E3 ligase are essential for mediating the inhibitory effect of high K intake on Kir4.1 and NCC in the DCT. Since basolateral Kir4.1 plays a key role in regulating WNK-SPAK-NCC in the DCT , understanding the role of Kir5.1 and Nedd4-2 in regulating Kir4.1 will provide an integrated view regarding the regulation of K and Na transport in the distal tubules.