Functional kinomics establishes a critical node of volume-sensitive cation-Cl - cotransporter regulation in the mammalian brain

Sci Rep. 2016 Oct 26;6:35986. doi: 10.1038/srep35986.


Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation - a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl--sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl- uptake and stimulation of KCC3-mediated Cl- extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought "Cl-/volume-sensitive kinase" of the cation-Cl- cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood-Brain Barrier
  • Brain / metabolism*
  • Cell Size
  • Chlorides / metabolism*
  • HEK293 Cells
  • Humans
  • Ion Transport
  • Mice
  • Mice, Knockout
  • Mice, Transgenic
  • Osmotic Pressure
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / deficiency
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteomics
  • RNA Interference
  • Sodium-Potassium-Chloride Symporters / metabolism
  • Solute Carrier Family 12, Member 2 / metabolism
  • Symporters / antagonists & inhibitors
  • Symporters / deficiency
  • Symporters / genetics
  • Symporters / metabolism*


  • Chlorides
  • SLC12A6 protein, human
  • Slc12a2 protein, mouse
  • Slc12a6 protein, mouse
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 2
  • Symporters
  • Stk39 protein, mouse
  • Protein-Serine-Threonine Kinases
  • WNK3 protein, human
  • Wnk3 protein, mouse