Molecular mechanisms of WNK-SPAK/OSR1 regulation of transepithelial ion transport in the Drosophila renal tubule

项目总结 跨上皮离子转运障碍是临床细胞外容量、血压和 电解质，但跨上皮离子转运的分子机制很难直接在 哺乳动物的肾单位。申请者的长期目标是更好地了解上皮细胞离子转运 与人类肾功能相关的机制，充分的分子细节，以定义新的治疗方法 战略。该应用程序的总体目标是确定由以下组成的激酶级联的调节因子 WNK(不含赖氨酸)和Spak/OSR1(Ste20相关的富含脯氨酸的丙氨酸激酶/氧化应激反应) 通过调节肾脏在钠、钾稳态中起重要作用的激酶 跨上皮离子转运。该应用建立在三个最新发现的基础上：CL-直接与WNK结合 抑制自磷酸化和激活的激活域；支架蛋白Mo25(小鼠蛋白 25/Cab39)增强Spak/OSR1活性，低钾饮食激活WNK-Spak/OSR1 发信号。中心假设是跨上皮细胞离子流量直接受转运离子(Cl-和 K)通过调制WNK-Spak/OSR1信号，而Mo25提供额外的调节控制。这个 理论基础是，对这些分子机制的更好理解将使设计新颖的 治疗与较少的偏离目标的影响。在强劲的初步数据指引下，核心假设将是 通过追求三个特定目标进行测试：1)确定氯离子和钾在WNK异构体调节中的作用 在跨上皮离子转运中；2)确定Mo25在运输上皮细胞WNK信号中的作用； (3)使用新开发的化学WNK抑制剂探索肾小管生理。这种方法是创新的 通过将从生物物理学研究中获得的基本分子见解与功能生理学 这些分子机制的作用，使用新开发的平台和工具来探索 运输上皮生物学。已经建立了检测方法，并在调查人员的 手，研究氯离子和钾在体外和苍蝇肾脏对果蝇和哺乳动物WNKS的调节 小管，并测量活小管内的氯离子，时间分辨率；以及测量跨上皮 转基因或药物治疗的小管中的离子流量。这项拟议的研究意义重大， 因为这有望促进对WNK-Spak/OSR1调控的分子机制的理解。 一种运输肾上皮的细胞。研究将确定：1)细胞内氯离子变化的速度有多快 WNK活性；2)WNK是否作为K感受器；3)Mo25在跨上皮离子转运中的作用。在……里面 此外，这些研究将进一步开发最近发现的药理WNK抑制剂，这将是一种 进一步探讨果蝇和哺乳动物WNK-Spak/OSR1信号生物学的有用工具 系统，并有可能成为未来开发治疗化合物的基础 容量超负荷、高血压和高钾血症。

PROJECT SUMMARY Disorders of transepithelial ion transport underlie clinical disorders of extracellular volume, blood pressure, and electrolytes, but molecular mechanisms of transepithelial ion transport are difficult to directly examine in the mammalian nephron. The applicants' long-term goal is to better understand epithelial ion transport mechanisms relevant to human kidney function, in sufficient molecular detail to define new therapeutic strategies. The overall objective of this application is to identify regulators of a kinase cascade, consisting of WNK (With No Lysine) and SPAK/OSR1 (Ste20-related proline alanine rich kinase/oxidative stress response) kinases, that plays an essential role in sodium and potassium homeostasis through the regulation of renal transepithelial ion transport. The application builds on three recent findings: Cl- directly binds to the WNK kinase domain to inhibit autophosphorylation and activation; the scaffold protein Mo25 (Mouse protein 25/Cab39) enhances the activity of SPAK/OSR1; and low potassium diet activates WNK-SPAK/OSR1 signaling. The central hypothesis is that transepithelial ion flux is directly regulated by transported ions (Cl- and K+) through modulation of WNK-SPAK/OSR1 signaling, while Mo25 provides additional regulatory control. The rationale is that better understanding of these molecular mechanisms will allow the design of novel therapeutics with fewer off-target effects. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the roles of Cl- and K+ in the regulation of WNK isoforms in transepithelial ion transport; 2) Determine the role of Mo25 in WNK signaling in a transporting epithelium; and 3) Probe tubule physiology using newly developed chemical WNK inhibitors. The approach is innovative by bridging fundamental molecular insights gained from biophysical studies, with the functional physiological roles of those molecular mechanisms, using newly developed platforms and tools to probe questions of transporting epithelium biology. Assays have been established, and demonstrated feasible in the investigators' hands, to examine regulation of Drosophila and mammalian WNKs by Cl- and K+ in vitro and in the fly renal tubule, and to measure intracellular Cl- in live tubules, with temporal resolution; and to measure transepithelial ion flux in genetically modified, or pharmacologically treated, tubules. The proposed research is significant, because it is expected to advance understanding of molecular mechanisms of WNK-SPAK/OSR1 regulation in a transporting renal epithelium. The studies will determine: 1) how quickly changes in intracellular Cl- change WNK activity; 2) whether WNKs act as K+ sensors; and 3) the role of Mo25 in transepithelial ion transport. In addition, these studies will further develop recently identified pharmacological WNK inhibitors, which will be a useful tool for further probing the biology of WNK-SPAK/OSR1 signaling in Drosophila and mammalian systems, and potentially serve as the basis for future development of therapeutic compounds for the treatment of volume overload, hypertension and hyperkalemia.