Control of the Renal WNK Signaling Pathway by Phase Transitions

无赖氨酸（WNK）激酶是普遍存在的阳离子氯共转运调节剂。致病 这些激酶的突变引起盐敏感性高血压、高钾血症和IV型肾小管酸中毒， 表明它们在肾小管功能中的重要性。尽管我们对WNK的理解有了进步 信号通路，基本问题仍然存在。最大的知识差距涉及机制 WNK激酶通过细胞体积变化被激活。最近，我们报道了“长”激酶- WNK 1的活性形式（L-WNK 1）在高渗细胞收缩期间在生物分子凝聚物内活化。 这些无膜液体状组装体通过大分子拥挤诱导的相分离形成。 这一过程绕过了由外渗引起的细胞内氯化物增加的抑制作用， 引发净离子流入和体积恢复。蛋白质相位行为在WNK信号传导中的重要性是 进一步得到我们在远曲小管（DCT）中的工作的支持。在这个肾单位片段中，KS-WNK 1，一个截短的 WNK 1亚型可能作为相变的支架，驱动特化的 称为WNK体的生物分子凝聚物。这些结构在低钾血症期间在DCT中形成， 存在与磷酸化依赖的激活噻嗪敏感的NaCl协同转运蛋白 NCC。这表明细胞外K+敏感，WNK相行为和远端肾单位之间的关系。 盐处理在这里，我们将测试WNK激酶经历相变以响应其功能的假设。 环境和放大离子运输。目标1将确定磷酸化依赖的电荷开关 L-WNK 1 C-末端的激活离子通量和体积恢复。目的2将研究细胞内氯如何 和拥挤诱导的相分离相互作用以控制WNK活性。目标3将确定DCT WNK车身 冷凝物是NCC激活所必需的，以及破坏WNK体是否逆转家族性 高钾血症性高血压。该提案结合了WNK两位领导人的互补专业知识 信令字段。MPI将使用多方面的创新方法，包括改变的小鼠模型， WNK身体功能，新开发的活细胞成像方法，以及果蝇Malpighian小管，一个易于处理的 和遗传可塑性的肾单位模型。WNK激酶作为生理功能的发现 拥挤的传感器标志着一个概念上的进步，它使新的思维和逻辑上可检验的假设成为可能， 透过凝聚生物学的透镜来观察。因此，我们希望从这些研究中获得的知识 将改变我们对上皮细胞体积调节和离子转运的理解，同时测试功能性 生物分子凝聚物对肾脏生理和疾病的贡献。

With-No-Lysine (WNK) kinases are ubiquitous regulators of cation chloride cotransport. Disease-causing mutations of these kinases cause salt-sensitive hypertension, hyperkalemia, and type IV renal tubular acidosis, indicating their importance in kidney tubular function. Despite advances in our understanding of the WNK signaling pathway, fundamental questions remain. The most significant knowledge gap concerns the mechanism by which WNK kinases are activated by cell volume changes. Recently, we reported that the “Long” kinase- active form of WNK1 (L-WNK1) activates within biomolecular condensates during hypertonic cell shrinkage. These membraneless liquid-like assemblies form via phase separation, induced by macromolecular crowding. This process bypasses the inhibitory effects of increased intracellular chloride caused by exosmosis, rapidly triggering net ion influx and volume recovery. The importance of protein phase behavior in WNK signaling is further supported by our work in distal convoluted tubule (DCT). In this nephron segment, KS-WNK1, a truncated WNK1 isoform that likely functions as a scaffold for phase transitions, drives the formation of specialized biomolecular condensates termed WNK bodies. These structures form in the DCT during hypokalemia, and their presence correlates with the phosphorylation-dependent activation of the thiazide-sensitive NaCl cotransporter NCC. This suggests a relationship between extracellular K+ sensing, WNK phase behavior, and distal nephron salt handling. Here, we will test the hypothesis that WNK kinases undergo phase transitions to respond to their environment and amplify ion transport. Aim 1 will determine how phosphorylation-dependent charge switching of the L-WNK1 C-terminus activates ion flux and volume recovery. Aim 2 will investigate how intracellular chloride and crowding-induced phase separation interact to control WNK activity. Aim 3 will determine if DCT WNK body condensates are necessary for NCC activation, and whether disrupting WNK bodies reverses Familial Hyperkalemic Hypertension. This proposal combines the complementary expertise of two leaders in the WNK signaling field. The MPIs will use a multifaceted and innovative approach that includes mouse models of altered WNK body function, newly developed live cell imaging methods, and Drosophila Malpighian tubule, a tractable and genetically malleable model of the nephron. The discovery that WNK kinases function as physiological crowding sensors marks a conceptual advance that has enabled fresh thinking and logical testable hypotheses, viewed through the lens of condensate biology. Thus, we expect that the knowledge gained from these studies will transform our understanding of epithelial cell volume regulation and ion transport, while testing the functional contributions of biomolecular condensates to kidney physiology and disease.