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

Abstract

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.

Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport

“…We have confirmed that in vitro both compounds potently suppress CCT domain binding to RFXV motifs, but that in cellular studies we observed that only STOCK1S-50699 but not STOCK2S-26016 suppressed SPAK/OSR1 and NKCC1 phosphorylation induced by hypotonic low chloride conditions [12, 52]. Consistent with STOCK1S-50699 and STOCK2S-26016 being selective, they did not inhibit the activity of 139 different protein kinases tested [102].…”

“…Yeast-2-hybrid experiments have originally demonstrated that a unique 90 amino acid domain, the conserved C-terminal (“CCT”) docking domain, of SPAK and OSR1 bind a conserved peptide motif of their downstream targets [8]. The motifs are RFXV/I in the N-terminus of NCC, NKCC1, and NKCC2 [3, 51], RFMV motif in the N-terminus of KCC2A and KCC3A [12, 52]. However, KCC1 and KCC4 have HFTV or NFTV motif in their N-terminus which did not show interaction with SPAK/OSR1 [8, 53].…”

Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential

“…These processes of RVI and RVD are examples that demonstrate how neuronal volume and Cl − movement are linked through CCCs [113,114]. In turn, CCCs are regulated by [Cl − ] i and cell volume [16,89].…”

Modulation of brain cation-Cl− cotransport via the SPAK kinase inhibitor ZT-1a

“…The electroneutral cation-Cl − cotransporters (CCCs) are secondary-active plasmalemmal ion transporters that utilize electrochemically favorable transmembrane gradients of Na + and/or K + , established by the ouabain-sensitive Na + , K + -ATPase to drive transport of Cl − (and Na + /K + ) into or out of cells. In epithelial cells under most physiological conditions (with the possible exception of choroid plexus), the Na + -driven CCCs NCC, NKCC1, and NKCC2 ("N(KCCs") [17][18][19][20][21][22] , function as Cl − importers, whereas the Na + -independent KCC1-4 ("KCCs") [23][24][25][26] function as Cl − exporters. These evolutionarily conserved transporters 27 are of particular importance in regulation of ion and water homeostasis in mammalian central nervous system (CNS) 28,29 .…”