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

Jinwei Zhang (Lead / Corresponding author), Geng Gao, Gulnaz Begum, Jinhua Wang, Arjun R. Khanna, Boris E. Shmukler, Gerrit M. Daubner, Paola De Los Heros, Paul Davies, Joby Varghese, Mohammad Iqbal H. Bhuiyan, Jinjing Duan, Jin Zhang, Daniel Duran, Seth L. Alper, Dandan Sun, Stephen J. Elledge, Dario R. Alessi, Kristopher T. Kahle (Lead / Corresponding author)

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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 Thr(991)/Thr(1048) 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 (Thr(203)/Thr(207)/Thr(212)), is also essential for the inhibitory phosphorylation of KCC3 (Thr(991)/Thr(1048)). 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.

Original languageEnglish
Article number35986
Pages (from-to)1-19
Number of pages19
JournalScientific Reports
Publication statusPublished - 26 Oct 2016


  • Chloride channels
  • Ion channel signalling
  • Kinases
  • Potassium channels


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