Regulation of WNK signaling by potassium and Mo25: structure, function and physiology

人类WNK(不含赖氨酸)激酶突变与高钾血症、高血压和慢性疾病相关 肾脏疾病。然而，尽管WNKS在肾脏的(病理)生理作用得到了广泛的表征 和肾外组织，令人惊讶的是，对WNK本身是如何调节的知之甚少。WNK 调节哺乳动物肾脏上皮细胞离子转运。申请者的长期目标是实现 从机制上理解与人类肾功能有关的上皮离子转运机制。氯化物 已知离子调节WNKS。本应用程序的总体目标是定义和理解新的 WNK的调节机制。此续订申请建立在当前基础上的三大进步之上 资助金。首先，钾通过不依赖氯的方式抑制果蝇和哺乳动物的WNKS 机械装置。其次，支架蛋白Mo25(Mouse Protein 25/Cab39)是WNK的重要调节因子 并激活WNKS，而不依赖于其对Spak(Ste20相关的富含Pro丙氨酸的Spak)的已知影响 激酶)和OSR1(氧化应激反应)激酶。第三，钾和钼对玉米生长发育的影响不同。 肾脏表达哺乳动物WNKS1、3和4。中心假说是钾和Mo25直接 调节WNK的活性，对哺乳动物的WNK亚型有不同的影响。以强劲的前期工作为指导 数据，将通过追求三个具体目标来检验中心假设：1)确定机制和 钾调节WNK的生理效应；2)阐明Mo25调节的新机制 3)钾对WNK异构体的不同调控的分子基础 和莫伊特25。这种方法是创新的，它利用生物物理和结构研究的见解来确定 利用独特的平台--马氏果蝇--调节上皮离子转运的分子机制 它拥有强大的分子遗传学工具和易于处理的生理读数。化验结果已经被 建立并在研究人员手中证明是可行的，以：1)确定WNK钾结合部位， 产生对钾不敏感的WNK突变体，并检测其对跨上皮离子转运的影响；2)确定 Mo25在体外和肾小管内如何调节WNK活性，并探讨Mo25与钾的相互作用 3)钾和Mo25对WNKS 1、3和4的调控差异。 成功完成拟议的研究将阐明最全面的机理理解 到目前为止，世界自然资源中心的监管工作取得了进展。这一点意义重大，因为描绘了这些WNK的重要性 各种(病理)生理环境中的调节机制，以及从以下方面获得的分子见解 这里提出的研究将允许开发有针对性的方法来治疗调节WNK 发信号。这在广泛的条件下产生了翻译影响，包括治疗颅脑功能障碍， 盐敏感型高血压、心脑血管疾病和肾脏疾病。

Mutations in human WNK (With No Lysine) kinases are associated with hyperkalemia, hypertension and chronic kidney disease. However, despite extensive characterization of (patho)physiological roles of WNKs in the kidney and extrarenal tissues, there is surprisingly little understanding of how WNKs themselves are regulated. WNKs regulate epithelial ion transport in the mammalian kidney. The applicants’ long-term goal is to achieve mechanistic understanding of epithelial ion transport mechanisms relevant to human kidney function. Chloride ion is known to regulate WNKs. The overall objective of this application is to define and understand new mechanisms of WNK regulation. This renewal application builds on three significant advances from the currently funded grant. First, potassium inhibits Drosophila and mammalian WNKs through chloride-independent mechanisms. Second, the scaffold protein Mo25 (Mouse protein 25/Cab39) is an important regulator of WNK signaling, and activates WNKs independent of its known effects on SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress response) kinases. Third, potassium and Mo25 have differential effects on the kidney-expressed mammalian WNKs 1, 3 and 4. The central hypothesis is that potassium and Mo25 directly regulate WNK kinase activity, with differential effects on mammalian WNK isoforms. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the mechanism and physiological consequences of WNK regulation by potassium; 2) Elucidate novel mechanisms of Mo25 regulation of WNK signaling; and 3) Determine the molecular basis for differential WNK isoform regulation by potassium and Mo25. The approach is innovative by leveraging insights from biophysical and structural studies to determine molecular mechanisms of epithelial ion transport regulation, using a unique platform, the Drosophila Malpighian tubule, that has powerful molecular genetic tools and tractable physiologic readouts. Assays have been established, and demonstrated feasible in the investigators’ hands, to: 1) identify WNK potassium binding sites, generate potassium-insensitive WNK mutants, and test their effects on transepithelial ion transport; 2) determine how Mo25 regulates WNK activity in vitro and in the tubule, and probe the interactions between Mo25, potassium and chloride in WNK regulation; and 3) test differences in potassium and Mo25 regulation of WNKs 1, 3 and 4. Successful completion of the proposed studies will elucidate the most comprehensive mechanistic understanding of WNK regulation achieved to date. This is significant, because delineation of the importance of these WNK regulatory mechanisms in various (patho)physiological contexts, together with the molecular insights gained from the studies proposed here, will allow the development of targeted approaches to therapeutically modulate WNK signaling. This has translational impact in a broad range of conditions, including the treatment of dyskalemias, salt-sensitive hypertension, cardio- and cerebrovascular disease, and kidney disease.