Cell volume recovers from hypertonicity-induced shrinkage via the process of regulatory volume increase (RVI). The sodium-potassium-2-chloride cotransporter NKCC1 was the first molecule identified as important in RVI (1) and is considered central to the process (2). NKCC1 regulation has been studied in the contexts of RVI and transepithelial transport (3–5). Two distinct signals, hypertonicity (low volume) and low intracellular chloride, control NKCC1 in these contexts. Both regulatory mechanisms were found to be phosphorylation dependent, leading to the hypothesis of a volume-and chlorideregulated protein kinase (6). Lytle and McManus (3) concluded that the volume and chloride signals are “mutually interdependent” and “converge on a single protein kinase.” The with-no-lysine (WNKs)[K] kinases, discovered by Cobb and colleagues (7), are proving to be the long sought-after kinases activated dually by hypertonicity and low intracellular chloride. Serra et al.(8) report the chloride channel leucine rich repeat containing 8 subunit A (LRRC8A) as an gene essential for RVI and show that it is essential for WNK activation. WNKs are activated by osmotic stressors (7, 9), and roles for WNKs in volume recovery have been demonstrated in human embryonic kidney 293 (HEK293) cells and in worms (10–12). WNKs are also activated by experimental maneuvers that lower intracellular chloride (9, 13). The discovery of LRRC8A coincides with an improved molecular-level understanding of how WNKs are regulated by chloride and osmotic stresses. Chloride binds and stabilizes an auto-inhibited dimeric configuration (14). Osmotic stress, mimicked in vitro with crowding agents (15), induces WNK1 and WNK3 to autophosphorylate (16). Crowding agents induce monomerization as measured by gel filtration and low-angle X-ray scattering. Overall, WNKs undergo a solvent-and chloride-driven conformational equilibrium between a Cl−-bound inactive state and a Cl−-unbound activation-competent state (16). Osmotic pressure exerts a demand on solvent to shift the equilibrium to the latter state (16, 17)(Fig. 1A). In support of these in vitro studies, mice carrying amino acid mutation of the chloride binding sites of WNK4 exhibit constitutive activation of the kinase (18). The data provide compelling support that WNKs are the volume-regulated kinases that sense intracellular chloride. An apparent conundrum remains, however; osmotic stress activates WNKs and NKCCs, but the intracellular chloride concentration rises as a result of shrinkage-induced water loss and from NKCC1-mediated chloride entry during RVI, which is expected to inhibit WNKs. In PNAS, Serra et al.(8) report a gene essential for hypertonic stress responses and thereby, may have resolved this long-standing question in cell volume regulation. The authors used the Toronto KO library, a large CRISPR-Cas9 guide RNA library (19), to identify genes essential for survival after hypertonic stress. The screen, conducted in HeLa cells (immortal cervical cancer cell line from Henrietta Lacks), identified the chloride channel LRRC8A (Swell1) as the most significant osmo-protective gene among nonlethal genes. Apparently, a chloride channel is involved in regulating intracellular chloride levels in the context of RVI. By reducing intracellular chloride, LRRC8A permits the osmotically induced activation. This occurs in symmetrical cells in the context of RVI but reflects the mechanism of chloride reduction in chloride transepithelial transport (20). It is interesting that many of the genes essential to cell volume recovery, NKCC1, WNKs, and p38 mitogen activated protein (MAP) kinase, did not appear in the Serra et al.(8 …