Chloride Sensing by WNK Kinases

WNK 激酶的氯离子传感

• 批准号: 8506799
• 负责人: ELIZABETH J. GOLDSMITH
• 金额: $ 27.83万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-18 至 2015-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8506799
• 关键词: Active Sites; Affect; Binding; Binding Sites; Biological Assay; Calorimetry; Cations; Cells; Chloride Ion; Chlorides; Crystallography; Docking; Drug Targeting; Enzymes; Head; Homeostasis; Hypertension; Ions; Learning; Length; Link; Lysine; Mammalian Cell; Mammals; Measurement; Measures; Phosphorylation; Phosphorylation Site; Phosphotransferases; Potassium; Protein Isoforms; Protocols documentation; Regulation; Regulatory Element; Sodium Chloride; Structure; Testing; X-Ray Crystallography; base; design; mutant; public health relevance; response; salt balance; salt sensitive; sensor

DESCRIPTION (provided by applicant): Chloride is a known regulator of ion transporters, yet chloride sensors that control transport have not been identified in mammals. We discovered that the WNK [with no lysine (K)] kinase, WNK1, known to be salt sensitive, is directly regulated by chloride, and found the chloride binding site in structural studies. Here we will determine whether one or all WNKs (isoforms 1, 2, 3, and 4) are regulated by chloride, and over what concentration ranges, using assays, thermal stability measurements and isothermal tritration calorimetry. We will determine how chloride affects the structure of the kinase domain of WNK1 through structural comparisons of chloride-bound and active WNK1, with existing crystals. WNKs are regulated by phosphorylation and autoinhibition. How the chloride inhibition relates to these additional mechanisms will be assessed by assays and crystallography. Mutants affecting chloride binding will be designed and evaluated. The WNK-based chloride sensing mechanism will be tested in cells.

描述（由申请人提供）：氯化物是离子转运蛋白的已知调节剂，但是尚未在哺乳动物中鉴定出控制转运的氯化物传感器。我们发现WNK [没有赖氨酸（K）]激酶，WNK1（已知是盐敏感的）直接受氯化物的调节，并在结构研究中发现了氯化物结合位点。在这里，我们将确定一个或所有WNK（同工型1、2、3和4）是否受氯化物的调节，以及使用测定，热稳定性测量和等温静脉热量法的浓度范围。我们将通过现有晶体来确定氯化物如何通过氯化物结合和活性WNK1的结构比较来影响WNK1的激酶结构域的结构。 WNK受磷酸化和自身抑制的调节。氯化物抑制与这些其他机制的关系将通过测定和晶体学评估。将设计和评估影响氯化物结合的突变体。基于WNK的氯传感机制将在细胞中进行测试。