Regulation of Renal Maxi K Channel by WNK Kinase

描述（由申请人提供）： 本研究的目的是阐明WNK 4如何调节Maxi K通道功能的机制。WNK（不含赖氨酸（K））激酶是丝氨酸/苏氨酸激酶的一个亚家族（5）。WNK 1和WNK 4的突变导致假性醛固酮减少症II型（PHA II）。PHA II，也称为Gordon综合征，是一种常染色体显性遗传疾病，以高血压、高钾血症和代谢性酸中毒为特征（6）。新出现的证据表明，WNK激酶有助于一种新的信号通路，涉及调节离子转运蛋白和控制钠和钾稳态的通道。显示WNK 4 WT抑制肾外髓钾（ROMK）通道活性和表面表达，而WNK 4 PHA II引起突变体进一步增强对ROMK活性的抑制（7）。WNK 1 WT也显示出抑制ROMK活性，而肾脏特异性形式的WNK 1（KS-WNK 1）逆转WNK 1介导的ROMK抑制（8）。Maxi K，也称为BK或Slo，是远端肾单位中的另一种主要钾通道（9）。我们的初步数据表明，WNK 4 WT不仅显著抑制Maxi K通道活性和细胞表面表达，而且降低哺乳动物细胞中Maxi K的总蛋白表达。我们还发现，WNK 4死亡激酶突变体D321 A失去了对Maxi K通道活性的抑制作用，表明WNK 4对Maxi K通道的抑制作用是激酶依赖性的。我们还发现，WNK 4增强ERK 1/2磷酸化，而敲低WNK 4降低ERK 1/2磷酸化。MAPK抑制剂U 0126可逆转WNK 4介导的Maxi K蛋白表达抑制。这些数据表明，WNK 4通过MAPK信号通路影响Maxi K活性和蛋白表达。我们还发现WNK 4 WT与Maxi K相互作用，而WNK 4死激酶突变体D321 A保留与Maxi K的相互作用，但失去其对Maxi K活性的抑制作用。然而，WNK 4是否需要结合Maxi K以调节Maxi K通道活性仍有待阐明。本申请的中心假设是WNK 4激酶通过影响Maxi K蛋白加工或通过改变Maxi K的磷酸化来调节Maxi K活性。为了检验这一假设，将提出以下具体目标：具体目标1：确定WNK激酶和Maxi K通道的哪些结构域负责它们的相互作用，并确定它们在肾小管细胞和天然组织中的共定位位置。具体目标2：确定WNK 4对Maxi K蛋白加工的抑制作用的机制。具体目标3：确定WNK 4如何通过磷酸化依赖性机制影响Maxi K活性。具体目标4：确定饮食钾变化和过表达WNK 4对小鼠肾钾排泄和Maxi K蛋白丰度的影响。我们将应用膜片钳技术、细胞生物学、分子生物学、蛋白质生物化学和动物体内研究等方法进行实验。进一步探讨WNK对Maxi K通道功能的影响，阐明WNK激酶突变引起的PHA II的病理生理机制，将为该研究领域中WNK激酶信号对钾通道活性和钾分泌的调控提供新的视角。

DESCRIPTION (provided by applicant): The aim of this proposal is to elucidate the mechanism of how WNK4 regulates Maxi K channel function. WNK (with no lysine (K)) kinase is a subfamily of serine/threonine kinases (5). Mutations in WNK1 and WNK4 result in pseudohypoaldosteronism type II (PHA II). PHA II, also referred to as Gordon syndrome, is an autosomal dominant disorder, characterized by hypertension, hyperkalemia, and metabolic acidosis (6). Emerging evidence indicates that WNK kinase contributes to a novel signaling pathway involving the regulation of ion transporters and channels that control sodium and potassium homeostasis. WNK4 WT was shown to inhibit renal outer medullary potassium (ROMK) channel activity and surface expression, whereas WNK4 PHA II- causing mutant further enhances the inhibition of ROMK activity (7). WNK1 WT was also shown to inhibit ROMK activity, whereas a kidney specific form of WNK1 (KS-WNK1) reverses the WNK1-mediated inhibition of ROMK (8). Maxi K, also referred as to BK or Slo, is the other major potassium channel in the distal nephron (9). Our preliminary data show that WNK4 WT not only significantly inhibits Maxi K channel activity and cell surface expression, but also reduces the total protein expression of Maxi K in mammalian cells. We also showed that WNK4 dead-kinase mutant D321A loses its inhibitory effect on Maxi K channel activity, indicating that the inhitory effect of WNK4 on Maxi K is kinase-dependent. We also found that WNK4 enhanced ERK1/2 phosphorylation, whereas knock-down of WNK4 reduced ERK1/2 phosphorylation. U0126, a MAPK inhibitor, reversed WNK4-mediated inhibition of Maxi K protein expression. These data indicated that WNK4 affects Maxi K activity and protein expression through a MAPK signaling pathway. We also showed that WNK4 WT interacts with Maxi K, whereas WNK4 dead-kinase mutant, D321A, retains interact with Maxi K while losing its inhibitory effect on Maxi K activity. However, it remains to be elucidated whether or not it is necessary for WNK4 to bind Maxi K in order to modulate Maxi K channel activity. The central hypothesis of this application is that WNK4 kinase modulates Maxi K activity either through affecting Maxi K protein processing or through altering the phosphorylation of Maxi K. To test this hypothesis, the following specific aims will be proposed: Specific Aim 1: Identify which domains of WNK kinase and Maxi K channel are responsible for their interaction and determine where they are colocalized in renal tubule cells and native tissue. Specific Aim 2: Determine the mechanism of the inhibitory effect of WNK4 on Maxi K protein processing. Specific Aim 3: Determine how WNK4 affects Maxi K activity through a phosphorylation dependent mechanism. Specific Aim 4: Determine the effects of dietary potassium changes and over-expressed WNK4 on renal potassium excretions and Maxi K protein abundances in mice. We will apply the methodologies of patch-clamp technique, cell biology, molecular biology, protein biochemistry and in vivo animal study to perform the proposed experiments. Further exploration of how WNK affects Maxi K channel function as well as the elucidation of the underlying pathophysiologic mechanism of PHA II caused by mutations of WNK kinases will provide a novel view on the regulation of the potassium channel activity and K secretion involving WNK kinase signaling in this research field.