title: WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway.
abstract: SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.
authors.affiliation: Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan, Mexico City, 14000, Mexico.
authors.forename: Paola
authors.initials: P
authors.lastname: de Los Heros
authors.forename: Kristopher T
authors.initials: KT
authors.lastname: Kahle
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.forename: Norma A
authors.initials: NA
authors.lastname: Bobadilla
authors.forename: Norma
authors.initials: N
authors.lastname: Vázquez
authors.forename: Pedro
authors.initials: P
authors.lastname: San Cristobal
authors.forename: David B
authors.initials: DB
authors.lastname: Mount
authors.forename: Richard P
authors.initials: RP
authors.lastname: Lifton
authors.forename: Steven C
authors.initials: SC
authors.lastname: Hebert
authors.forename: Gerardo
authors.initials: G
authors.lastname: Gamba
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007038:Hypotonic Solutions; D007700:Kinetics; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D000096922:K Cl- Cotransporters
chemical_list: D007038:Hypotonic Solutions; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D027981:Symporters; D000096922:K Cl- Cotransporters