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

钾和 Mo25 对 WNK 信号传导的调节：结构、功能和生理学

基本信息

批准号：
10298458
负责人：
AYLIN RACHEL RODAN
金额：
$ 47.48万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-13 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298458
关键词：
Alanine Animal Model Binding Binding Sites Biochemical Biological Assay Biophysics Blood Calorimetry Cerebrovascular Disorders Cesium Chloride Ion Chlorides Chronic Kidney Failure Crystallization Data Development Drosophila genus Drosophila melanogaster Epithelial Equilibrium Funding Future Goals Grant Hand Health Human Hypertension In Vitro Ion Transport Kidney Kidney Diseases Lead Lysine Malpighian Tubules Maps Measures Methods Modeling Molecular Molecular Conformation Molecular Genetics Morbidity - disease rate Mouse Protein Mutagenesis Mutation Organ Outcome Oxidative Stress Pathologic Pathway interactions Phosphotransferases Physiological Physiology Positioning Attribute Potassium Potassium Chloride Process Proline Protein Isoforms Publishing Regulation Renal function Research Research Personnel Roentgen Rays Role Scaffolding Protein Signal Pathway Signal Transduction Structure Testing Therapeutic Tissues Titrations adverse outcome base biological adaptation to stress collaborative approach conformer design dimer experience human model hyperkalemia in vitro activity in vivo inhibitor/antagonist innovation insight monomer mortality mutant new therapeutic target novel novel therapeutic intervention paralogous gene post stroke salt sensitive hypertension side effect tool translational impact

项目摘要

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.

人类 WNK（无赖氨酸）激酶突变与高钾血症、高血压和慢性然而，尽管 WNK 在肾脏中的（病理）生理作用已得到广泛表征。令人惊讶的是，对于 WNK 本身如何受到调节，人们却知之甚少。调节哺乳动物肾脏中的上皮离子转运。申请人的长期目标是实现。与人类肾功能相关的上皮离子转运机制的机制理解。众所周知，离子可以调节 WNK。该应用的总体目标是定义和理解新的。 WNK 监管机制的更新应用建立在当前的三个重大进步之上。首先，钾通过不依赖于氯的方式抑制果蝇和哺乳动物的 WNK。其次，支架蛋白Mo25（小鼠蛋白25/Cab39）是WNK的重要调节因子。信号传导，并激活 WNK，独立于其对 SPAK（Ste20 相关富含脯氨酸丙氨酸）的已知影响第三，钾和Mo25对肾脏表达的哺乳动物 WNK 1、3 和 4。中心假设是钾和 Mo25 直接调节 WNK 激酶活性，对哺乳动物 WNK 亚型有不同的影响。数据，中心假设将通过追求三个具体目标来检验：1）确定机制和钾调节 WNK 的生理后果；2) 阐明 Mo25 调节的新机制 WNK 信号传导的机制；以及 3) 确定钾对 WNK 亚型差异调节的分子基础该方法是创新的，它利用生物物理和结构研究的见解来确定。上皮离子转运调节的分子机制，使用独特的平台，果蝇马尔皮基安肾小管，具有强大的分子遗传工具和易于处理的生理检测。已在研究人员手中建立并证明可行：1）识别 WNK 钾结合位点，产生钾不敏感的WNK突变体，并测试它们对跨上皮离子转运的影响；2)确定 Mo25 如何在体外和肾小管中调节 WNK 活性，并探讨 Mo25 与钾之间的相互作用和氯在 WNK 调节中的作用；3) 测试 WNK 1、3 和 4 中钾和 Mo25 调节的差异。成功完成拟议的研究将阐明最全面的机制理解迄今为止所实现的 WNK 监管意义重大，因为这些 WNK 的重要性得到了界定。各种（病理）生理背景下的调节机制，以及从中获得的分子见解这里提出的研究将有助于开发治疗性调节 WNK 的靶向方法这对多种疾病都有转化影响，包括血钾障碍的治疗，盐敏感性高血压、心脑血管疾病、肾脏疾病。