The WNK-SPAK/OSR1 pathway: master regulator of cation-chloride cotransporters

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordThe WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress-responsive kinase 1). The WNK family senses changes in intracellular Cl(-) concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na(+)), potassium (K(+)), and chloride (Cl(-)) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl(-)-importing, Na(+)-driven CCCs or inhibit the Cl(-)-extruding, K(+)-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K(+) secretion to lower blood pressure while maintaining serum K(+). In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl(-) extrusion and promote γ-aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K(+)-sparing blood pressure-lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism.D.R.A. research in this area is supported by the Medical Research Council and the Wellcome Trust [grant number 091415] and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer). K.T.K. is supported by the Manton Center for Orphan Diseases at Children's Hospital Boston at Harvard Medical School, and the Harvard/MIT Joint Research Grants Program in Basic Neuroscience

GABAA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl--sensitive WNK1 kinase

This is the final version of the article. Available from the publisher via the DOI in this record.There is another ORE record for this publication: http://hdl.handle.net/10871/33406The K+-Cl-co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAAreceptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl-]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAAreceptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl-as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl--sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl-]ito GABAAR activity.This work was supported in part by Inserm, Sorbonne Université-UPMC, as well as the Fondation pour la Recherche Médicale (Equipe FRM DEQ20140329539 to J.C.P.), the Human Frontier Science Program (RGP0022/2013 to J.C.P.) and the Fondation pour la Recherche sur le Cerveau (to S.L.). Equipment at the IFM was also supported by DIM NeRF from Région Ile-de-France and by the FRC/Rotary ‘Espoir en tête’. M.H. was the recipient of a doctoral fellowship from the Université Pierre and Marie Curie, as well as from Bio-Psy Laboratory of excellence. K.T.K. is supported by the National Institutes of Health, the Simons Foundation, and the March of Dimes Foundation Basil O’Connor Award. The Poncer/Lévi lab is affiliated with the Paris School of Neuroscience (ENP) and the Bio-Psy Laboratory of excellence

Recessive Inheritance of Congenital Hydrocephalus With Other Structural Brain Abnormalities Caused by Compound Heterozygous Mutations in ATP1A3

Background: ATP1A3 encodes the α3 subunit of the Na+/K+ ATPase, a fundamental ion-transporting enzyme. Primarily expressed in neurons, ATP1A3 is mutated in several autosomal dominant neurological diseases. To our knowledge, damaging recessive genotypes in ATP1A3 have never been associated with any human disease. Atp1a3 deficiency in zebrafish results in hydrocephalus; however, no known association exists between ATP1A3 and human congenital hydrocephalus (CH). / Methods: We utilized whole-exome sequencing (WES), bioinformatics, and computational modeling to identify and characterize novel ATP1A3 mutations in a patient with CH. We performed immunohistochemical studies using mouse embryonic brain tissues to characterize Atp1a3 expression during brain development. / Results: We identified two germline mutations in ATP1A3 (p. Arg19Cys and p.Arg463Cys), each of which was inherited from one of the patient’s unaffected parents, in a single patient with severe obstructive CH due to aqueductal stenosis, along with open schizencephaly, type 1 Chiari malformation, and dysgenesis of the corpus callosum. Both mutations are predicted to be highly deleterious and impair protein stability. Immunohistochemical studies demonstrate robust Atp1a3 expression in neural stem cells (NSCs), differentiated neurons, and choroid plexus of the mouse embryonic brain. / Conclusion: These data provide the first evidence of a recessive human phenotype associated with mutations in ATP1A3, and implicate impaired Na+/K+ ATPase function in the pathogenesis of CH

Proteomics: in pursuit of effective traumatic brain injury therapeutics

Effective traumatic brain injury (TBI) therapeutics remain stubbornly elusive. Efforts in the field have been challenged by the heterogeneity of clinical TBI, with greater complexity among underlying molecular phenotypes than initially conceived. Future research must confront the multitude of factors comprising this heterogeneity, representing a big data challenge befitting the coming informatics age. Proteomics is poised to serve a central role in prescriptive therapeutic development, as it offers an efficient endpoint within which to assess post-TBI biochemistry. We examine rationale for multifactor TBI proteomic studies and the particular importance of temporal profiling in defining biochemical sequences and guiding therapeutic development. Lastly, we offer perspective on repurposing biofluid proteomics to develop theragnostic assays with which to prescribe, monitor and assess pharmaceutics for improved translation and outcome for TBI patients

Opposite temperature effect on transport activity of KCC2/KCC4 and N(K)CCs in HEK-293 cells

<p>Abstract</p> <p>Background</p> <p>Cation chloride cotransporters play essential roles in many physiological processes such as volume regulation, transepithelial salt transport and setting the intracellular chloride concentration in neurons. They consist mainly of the inward transporters NCC, NKCC1, and NKCC2, and the outward transporters KCC1 to KCC4. To gain insight into regulatory and structure-function relationships, precise determination of their activity is required. Frequently, these analyses are performed in HEK-293 cells. Recently the activity of the inward transporters NKCC1 and NCC was shown to increase with temperature in these cells. However, the temperature effect on KCCs remains largely unknown.</p> <p>Findings</p> <p>Here, we determined the temperature effect on KCC2 and KCC4 transport activity in HEK-293 cells. Both transporters demonstrated significantly higher transport activity (2.5 fold for KCC2 and 3.3 fold for KCC4) after pre-incubation at room temperature compared to 37°C.</p> <p>Conclusions</p> <p>These data identify a reciprocal temperature dependence of cation chloride inward and outward cotransporters in HEK-293 cells. Thus, lower temperature should be used for functional characterization of KCC2 and KCC4 and higher temperatures for N(K)CCs in heterologous mammalian expression systems. Furthermore, if this reciprocal effect also applies to neurons, the action of inhibitory neurotransmitters might be more affected by changes in temperature than previously thought.</p

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy

Role of SPAK-NKCC1 signaling cascade in the choroid plexus blood-CSF barrier damage after stroke.

This is the final version. Available from BMC via the DOI in this record. Availability of data and materials: Supporting data and information about used material are available from the corresponding author on reasonable request.BACKGROUND: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (SPAK). METHODS: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined. RESULTS: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. CONCLUSION: Stroke-induced pathological stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.National Institutes of HealthVeterans AdministrationVA Research Career Scientist awardUPMC Endowed Chair professorship for Brain Disorders Researc

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

This is the final version of the article. Available from the publisher via the DOI in this record.There is another record in ORE for this publication: http://hdl.handle.net/10871/33424Cell 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.We thank the excellent technical support of the MRC-Protein Phosphorylation and Ubiquitylation Unit (PPU) DNA Sequencing Service (coordinated by Nicholas Helps), the MRC-PPU tissue culture team (coordinated by Laura Fin), the Division of Signal Transduction Therapy (DSTT) antibody purification teams (coordinated by Hilary McLauchlan and James Hastie). We are grateful to the MRC PPU Proteomics facility (coordinated by David Campbell, Robert Gourlay and Joby Varghese). We thank for support the Medical Research Council (MC_UU_12016/2; DRA) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer; DRA). We thank Thomas J. Jentsch (Max-Delbrück-Centrum für Molekulare Medizin) for providing the KCC1/3 double KO mice and his reading of this manuscript. We thank Nathaniel Grey (Harvard) for providing the kinase inhibitor library used in this study (NIH LINCS Program grant U54HL127365). This work was also supported by a Harvard-MIT Neuroscience Grant (to KTK/SJE)