Chloride Sensing by WNK Kinases

描述(申请人提供)：氯离子是已知的离子转运蛋白的调节器，但在哺乳动物中还没有发现控制转运蛋白的氯离子传感器。我们发现盐敏感的WNK[不含赖氨酸(K)]激酶WNK1直接受氯的调节，并在结构研究中发现了氯结合部位。在这里，我们将使用分析、热稳定性测量和等温滴定量热法来确定是否有一个或所有WNK(异构体1、2、3和4)受氯离子调节，以及在什么浓度范围内。我们将通过氯结合和活性WNK1与现有晶体的结构比较来确定氯如何影响WNK1的激活域的结构。WNKs受磷酸化和自身抑制的调节。氯的抑制如何与这些额外的机制有关，将通过分析和结晶学进行评估。将设计和评估影响氯结合的突变体。基于WNK的氯离子传感机制将在细胞中进行测试。

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.