Regulation of Renal Maxi K Channel by WNK Kinase

WNK 激酶对肾 Maxi K 通道的调节

• 批准号: 8413384
• 负责人: HUI CAI
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413384
• 关键词: Affect; Anabolism; Animals; Binding; Cell Line; Cell surface; Cells; Cellular biology; Data; Diet; Dietary Potassium; Disease; Distal; Duct (organ) structure; Elderly; Electrolytes; Endocytosis; Equilibrium; Excretory function; Goals; High Prevalence; Homeostasis; Hypertension; In Vitro; Ions; KCNJ1 gene; Kidney; Lysine; MAPK Signaling Pathway Pathway; MAPK3 gene; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Metabolic acidosis; Methodology; Mitogen-Activated Protein Kinase Inhibitor; Molecular Biology; Mus; Mutate; Mutation; Nephrons; Patch-Clamp Techniques; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Population; Potassium; Potassium Channel; Process; Protein Biochemistry; Protein-Serine-Threonine Kinases; Proteins; Regulation; Renal tubule structure; Research; Role; Signal Pathway; Signal Transduction; Site; Sodium; Surface; Syndrome; Testing; Tissues; Type II Pseudohypoaldosteronism; U-0126; Veterans; blood pressure regulation; hyperkalemia; in vivo; knock-down; large-conductance calcium-activated potassium channels; mutant; novel; protein K; protein expression; research study

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.

描述(由申请人提供)： 本研究旨在阐明WNK4调控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。WNK1和WNK4基因突变导致假性醛固酮减少症II型(PHA II)。PHA II，也被称为Gordon综合征，是一种常染色体显性遗传性疾病，特征是高血压、高钾血症和代谢性酸中毒。新的证据表明，WNK激酶参与了一种新的信号通路，涉及调控钠钾动态平衡的离子转运体和通道。WNK4 WT可抑制肾外髓质钾通道的活性和表面表达，而WNK4 PHA II突变体则进一步增强了对ROMK活性的抑制作用。WNK1 WT也显示出抑制ROMK活性，而肾脏特异形式的WNK1(KS-WNK1)可逆转WNK1介导的ROMK抑制(8)。Maxi K，也被称为BK或Slo，是远端肾单位的另一主要钾通道(9)。我们的初步数据表明，WNK4 WT不仅显著抑制哺乳动物细胞的Maxi K通道活性和细胞表面表达，而且还降低了Maxi K的总蛋白表达。我们还发现WNK4死亡激酶突变体D321a失去了对Maxi K通道活性的抑制作用，这表明WNK4对Maxi K的抑制作用是依赖于激酶的。我们还发现WNK4促进了ERK1/2的磷酸化，而WNK4的敲除则降低了ERK1/2的磷酸化。MAPK抑制剂U0126可逆转WNK4对Maxi K蛋白表达的抑制作用。这些数据表明，WNK4通过MAPK信号通路影响Maxi K的活性和蛋白表达。我们还发现WNK4 WT与Maxi K相互作用，而WNK4死亡激酶突变体D321A保留了与Maxi K的相互作用，但失去了对Maxi K活性的抑制作用。然而，WNK4是否有必要与Maxi K结合以调节Maxi K通道活性仍有待阐明。这一应用的中心假设是WNK4激酶通过影响Maxi K蛋白的加工或通过改变Maxi K的磷酸化来调节Maxi K的活性。为了验证这一假说，将提出以下特定目标：特定目标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自然组织中的共存位置。具体目的2：确定WNK4对Maxi K蛋白加工的抑制作用机制。具体目标3：确定WNK4如何通过磷酸化依赖机制影响Maxi K活性。特定目标4：确定饮食中钾的变化和过表达的WNK4对小鼠肾脏钾排泄和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、蛋白质生物化学和体内动物研究的方法来进行拟议的实验。进一步探讨WNK对Maxi K通道功能的影响，阐明WNK激酶突变导致的PHA II的潜在病理生理机制，将为该领域涉及WNK信号的钾通道活性和K分泌的调控提供新的视角。