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Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

By Anke Hannemann and Peter W. Flatman


Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates

Topics: Research Article
Publisher: Public Library of Science
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Provided by: PubMed Central

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  1. (1998). A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry.
  2. (2002). A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter,
  3. (2005). Activation of ferret erythrocyte Na +-K +-2Cl 2 cotransport by deoxygenation.
  4. (2002). Activation of the Na-K-Cl cotransporter NKCC1 detected with a phosphospecific antibody.
  5. (1994). Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney.
  6. (2007). Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation.
  7. (2002). Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1).
  8. (2009). Changes in sodium pump expression dictate the effects of ouabain on cell growth.
  9. (1995). Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies.
  10. (1983). Effect of ouabain upon diuretic-sensitive K +-transport in cultured cells. Evidence for separate modes of operation of the transporter.
  11. (1982). Effects of ouabain and osmolarity on bumetanide-sensitive potassium transport in simian virustransformed 3T3 cells.
  12. Flatman PW (2003) Regulation of Na +-K +-2Cl 2 cotransport by threonine phosphorylation in ferret red cells.
  13. (2002). Functional and molecular characterization of the shark renal Na-K-Cl cotransporter: novel aspects.
  14. (2009). Functional expression of the Na-K-2Cl cotransporter NKCC2 in mammalian cells fails to confirm the dominant-negative effect of the AF splice variant.
  15. (2006). Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1.
  16. (1997). Ion and bumetanide binding by the Na-K-Cl cotransporter.
  17. (2001). Modulation of ion transport by direct targeting of protein phosphatase type I to the Na-K-Cl cotransporter.
  18. (2010). Molecular determinants of hyperosmotically activated
  19. (2005). Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters.
  20. (2010). Multiple pathways for Protein Phosphatase 1 (PP1) regulation of Na-K-2Cl cotransporter (NKCC1) function.
  21. (2003). PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1).
  22. (2002). Phosphorylation of the salivary Na +-K +-2Cl 2 cotransporter.
  23. (2009). Physiological roles of endogenous ouabain in normal rats.
  24. (2005). Regulation of erythrocyte Na-K2Cl cotransport by threonine phosphorylation.
  25. (1999). Regulation of Na +-K +-2Cl 2 cotransport by protein phosphorylation in ferret erythrocytes.
  26. (2002). Regulation of Na-K-2Cl cotransport by phosphorylation and protein-protein interactions.
  27. (2008). Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases.
  28. (1996). Regulatory phosphorylation of the secretory NaK-Cl cotransporter: modulation by cytoplasmic Cl.
  29. (2005). Regulatory phosphorylation sites in the NH2 terminus of the renal Na-K-Cl cotransporter (NKCC2).
  30. (2010). Role of the WNK-activated SPAK kinase in regulating blood pressure.
  31. (2003). Short-term stimulation of the renal Na-K-Cl cotransporter (NKCC2) by vasopressin involves phosphorylation and membrane translocation of the protein.
  32. (2000). Sodium-potassium-chloride cotransport.
  33. (2008). SPAK and OSR1: STE20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells.
  34. (1996). Stimulation of Na +-K +-2Cl cotransport in rat medullary thick ascending limb by dopamine.
  35. (1977). The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres.
  36. (2008). The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway.
  37. (2007). The selectivity of protein kinase inhibitors: a further update.
  38. (1995). Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport.
  39. (2005). WNK1 Regulates Phosphorylation of Cation-Chloride-coupled Cotransporters via the STE20-related Kinases, SPAK and OSR1.