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WNK1 Regulates Cell Volume via Crowding‐Induced Phase Transitions

When challenged by hypertonicity, dehydrated cells recover their volume though regulatory volume increase (RVI). This process is thought to be initiated by an undefined intracellular sensing and transduction mechanism that responds to macromolecular crowding (Parker JC AJP 1993). In many cell types, RVI is mediated by the bumetanide‐sensitive cotransporter NKCC1, which is phosphorylated and activated by the WNK‐SPAK/OSR1 pathway. Reconciling the mechanism of hypertonic stress‐induced activation of NKCC1 has been challenging, as upstream WNK kinases such as WNK1, though required for bumetanide‐sensitive RVI (Roy A AJP 2015), should be inactivated by the strengthened intracellular chloride concentration caused by cell shrinkage (Piala AT Sci Signaling 2014). We describe our efforts to unravel this paradox using a combination of high‐speed live cell and fluorescence lifetime imaging, correlative light electron microscopy, gene editing, and biochemical approaches in cells. During hyperosmotic cell shrinkage and macromolecular crowding caused by reduced cell water, WNK1 dynamically concentrates into membraneless liquid droplets. These changes in WNK1 localization occur at native levels of expression and are visible within physiologic range (25 mOsm above isotonicity), triggering a cascade of condensed WNK1 autoactivation, droplet fusion, cytosolic exchange, and downstream SPAK/OSR1/NKCC1 phosphorylation within a 15 minute bumetanide‐sensitive RVI time course. When cells are rapidly switched from hypertonic to hypotonic media, the WNK1 condensates quickly dissipate, confirming their reversible nature and association with physiologic changes in cell size and intracellular crowding. An optogenetic screening assay that employs the PHR domain of Cry2 to trigger light‐induced crowding and phase separation revealed that the formation of WNK1 condensates requires its large intrinsically disordered C‐terminus, whose strong regional disorder tendency is highly conserved across evolution despite poor sequence homology. This disorder‐encoded phase behavior allows WNK kinases to react to a crowded cytosol, bypass the ionic strength dilemma, and restore cell volume via mass action. Thus, our findings indicate that in addition to their well‐appreciated role as chloride sensors, WNK kinases are molecular crowding sensors that phase separate to coordinate a conserved hyperosmotic stress response. While this evolutionarily conserved mechanism appears to be critical for WNK kinases to sense cell shrinkage and amplify the NKCC1 RVI signal, we propose that it also impacts diverse WNK‐dependent signaling processes important in mammalian epithelial and neuronal physiology.