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

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 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 and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-duanyu1842K 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-duanyu1842K 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.

KEYWORDS: {{ getKeywords(articleDetailText.words) }}