Comparison of WNK1 and WNK3 Phase Behavior, Kinase Activity, and Volume Regulatory Function

During hypertonic stress, With-No-Lysine kinase 1 (WNK1) phase separates, forming biomolecular condensates that mediate cell volume recovery in response to crowding induced by cell shrinkage. Within the condensed phase, WNK kinases activate SPAK and OSR1, downstream effector kinases that directly phosphorylate NKCC and KCC cation chloride cotransporters at the plasma membrane. This results in net ion influx and volume recovery, a process termed regulatory volume increase (RVI). Prior studies have suggested that the brain-expressed paralog WNK3 may activate RVI more strongly than the more ancient and ubiquitously expressed WNK1. However, whether WNK3 drives the formation of signaling condensates, and how this might influence its ability to control cell volume is unclear. We hypothesized that relative to WNK1, WNK3 functions as a more potent cell volume regulator due to its intrinsic phase separation properties. To test this, we expressed fluorescent protein-tagged mammalian WNK1 and WNK3 constructs in a CRISPR-edited double knockout HEK293 cell line lacking background WNK1 and WNK3 expression (DKO cells), and compared their expression, activity, phase behavior, and function via live cell imaging and western blotting. In an equimolar plasmid transfection study, steady state WNK3 abundance was 342% greater than WNK1, suggesting that the WNK3 protein is inherently more stable. When transfection levels were normalized for equal protein expression, WNK3 formed larger condensates than WNK1 in response to hypertonic stress. In a subset of WNK3-expressing cells, condensates were present under baseline isotonic conditions, suggesting that WNK3 exhibits enhanced crowding sensitivity relative to WNK1. Biochemical analyses matched for protein expression also confirmed higher levels of phosphorylated SPAK and OSR1 in WNK3-expressing cells under both isotonic and hypertonic conditions. 4D live-cell imaging of the RVI response in cells subjected to 50mOsm sorbitol demonstrated that WNK3 mediates cell volume recovery more effectively than WNK1. Surprisingly, however, live cell imaging studies of stress-induced phase behavior revealed that WNK1 recruits SPAK to condensates more efficiently than WNK3. DKO cells expressing exogenous WNK3, SPAK, and WNK1 exhibited enhanced recruitment of SPAK to condensates compared to cells transfected with WNK3 and SPAK alone. Together, these findings establish WNK3 as a potent regulator of cell volume recovery, with distinct condensate dynamics and signaling mechanisms compared to WNK1. While WNK3 recruits SPAK to condensates less effectively than WNK1, its protein stability and enhanced crowding-sensitive phase behavior likely contribute to its strong volume regulatory potency. NIH R01DK098145, R01DK119252, U54DK137329, TL1DK143271 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.