WNK3 and WNK4 Exhibit Opposite Sensibility for Cell Volume and Intracellular Chloride Concentration
Authors: Pacheco‐Alvarez Diana , Carrillo‐Pérez Diego Luis , Mercado Adriana , Leyva‐Ríos Karla , Moreno Erika , Vazquez Norma , Gamba Gerardo 
Published: June 2021

BackgroundThe intracellular chloride concentration [Cl−]i and cell volume modulates the activity of the electroneutral cation‐coupled chloride cotransporters (CCCs) such as the NKCCs and the KCCs. Cell shrinkage or reduction of [Cl−]i induce phosphorylation of CCCs, which activates NKCCs and inhibits KCCs, promoting ion influx, while cell swelling or increase of [Cl−]i induces dephosphorylation, that inhibits NKCCs and activates KCCs, stimulating ion efflux. We have shown that the effect of [Cl−]i towards CCCs is traduced by the WNK1 or WNK4 kinases. However, cell volume and [Cl−]i moves in opposite directions. Cell shrinkage increases [Cl−]i and cell swelling decreases [Cl−]i. Thus, the effect of cell volume towards CCCs must be traduced by a different kinase. Because WNK3 bypasses the tonicity requirements for regulation of the CCCs (de Los Heros, PNAS 2006), we tested the hypothesis that WNK3 is sensitive to cell volume, rather than to the [Cl−]i.MethodsXenopus oocytes were microinjected with the Na‐Cl cotransporter (NCC) or the K‐Cl cotransporter (KCC4) cRNA alone or together with WNK3 or WNK4 wild type, or mutants in which catalytic activity and/or the chloride‐binding site has been eliminated (WNK3‐DA; WNK3‐L295/297F; WNK3‐DA‐LLFF; WNK4‐DA; WNK4‐L322F) or with chimeric constructs obtained by swapping the amino, kinase or carboxyl terminal domains between WNK3 and WNK4. The effect of [Cl−]i or extracellular tonicity on WNKs effects on NCC or KCC4 was assessed by tracer 22Na+ or 86Rb+ uptake, respectively. The effect on extracellular tonicity on WNKs activity was assessed as WNK phosphorylation at the activating serine of the T‐loop with specific antibodies by Western blot, as a surrogate of activity.ResultsDepletion of [Cl−]i increases the activity of WNK4 and thus increases the NCC activity. Elimination of WNK4 chloride‐binding site turned it into a constitutively active kinase toward NCC. In contrast the degree of activation of NCC by WNK3 was not affected by reduction of [Cl−]i and elimination of the chloride‐binding site in WNK3 had no effect on the activity of the kinase. Compared to isotonic condition, WNK3 phosphorylation significantly diminished or increased by 50% in hypotonic or hypertonic conditions, respectively. The phosphorylation of WNK4 was not affected by similar changes in tonicity. Chimeric proteins between WNK3 and WNK4 revealed that the carboxyl terminal domain modulates chloride affinity in WNKs. That is, swapping the WNK4 carboxyl terminal domain into WNK3 amino and kinase domain rendered this construct sensitive to changes in the [Cl−]i.DiscussionWe previously demonstrated that WNK4 is sensitive to [Cl−]i (Bazúa‐Valenti, JASN 2015). Here we show that WNK3 is not sensitive to changes of the [Cl−]i, but is modulated by changes in cell volume. In addition, WNK3, but not WNK4, phosphorylation was sensitive to changes in cell volume. Hypotonicity reduces WNK3 phosphorylation, while hypertonicity increased it. Thus, we propose that while WNK4 is a chloride sensitive kinase, WNK3 is a volume sensitive kinase, modulating the effect of cell volume changes towards CCCs activity.Support or Funding InformationFronteras de la Ciencia Grant No. 23 from Conacyt to GGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.