Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways

J Cell Sci. 2011 Mar 1;124(Pt 5):789-800. doi: 10.1242/jcs.077230.


Ion cotransporters, such as the Na(+)/Cl(-) cotransporter (NCC), control renal salt re-absorption and are regulated by the WNK-signalling pathway, which is over-stimulated in patients suffering from Gordon's hypertension syndrome. Here, we study the regulation of the NKCC2 (SLC12A1) ion cotransporter that contributes towards ~25% of renal salt re-absorption and is inhibited by loop-diuretic hypertensive drugs. We demonstrate that hypotonic low-chloride conditions that activate the WNK1-SPAK and OSR1 pathway promote phosphorylation of NKCC2 isoforms (A, B and F) at five residues (Ser91, Thr95, Thr100, Thr105 and Ser130). We establish that the SPAK and OSR1 kinases activated by WNK interact with an RFQV motif on NKCC2 and directly phosphorylate Thr95, Thr100, Thr105 and, possibly, Ser91. Our data indicate that a SPAK-OSR1-independent kinase, perhaps AMP-activated protein kinase (AMPK), phosphorylates Ser130 and that phosphorylation of Thr105 and Ser130 plays the most important roles in stimulating NKCC2 activity. In contrast with NCC, whose membrane translocation is triggered by SPAK-OSR1 phosphorylation, NKCC2 appears to be constitutively at the membrane. Our findings provide new insights into how NKCC2 is regulated and suggest that inhibitors of SPAK and/or OSR1 for the treatment of hypertension would be therapeutically distinct from thiazide or loop diuretics, as they would suppress the activity of both NCC and NKCC2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism
  • Amino Acid Sequence
  • Animals
  • Cell Membrane / metabolism
  • HEK293 Cells
  • Humans
  • Ions / metabolism
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Sequence Alignment
  • Serine / metabolism
  • Signal Transduction / physiology
  • Sodium-Potassium-Chloride Symporters / genetics
  • Sodium-Potassium-Chloride Symporters / metabolism*
  • Solute Carrier Family 12, Member 1
  • Threonine / metabolism


  • Ions
  • Protein Isoforms
  • SLC12A1 protein, human
  • Slc12a1 protein, mouse
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 1
  • Threonine
  • Serine
  • OXSR1 protein, human
  • Protein Serine-Threonine Kinases
  • STK39 protein, human
  • AMP-Activated Protein Kinases