Chloride Sensing by WNK Kinases

描述（由申请人提供）：氯化物是一种已知的离子转运体调节剂，但尚未在哺乳动物中发现控制转运的氯化物传感器。我们发现WNK[无赖氨酸(K)]激酶，WNK1，已知对盐敏感，直接受氯化物调节，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的wnk（异构体1、2、3和4）是否受氯化物调节，以及在什么浓度范围内，使用测定，热稳定性测量和等温滴定量热法。我们将通过氯结合和活性WNK1与现有晶体的结构比较，确定氯如何影响WNK1激酶结构域的结构。WNKs受磷酸化和自抑制调控。氯化物抑制作用与这些附加机制的关系将通过测定和结晶学来评估。将设计和评估影响氯化物结合的突变体。基于wnk的氯化物感应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.