The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK

Biochem Biophys Res Commun. 2008 Oct 31;375(4):651-4. doi: 10.1016/j.bbrc.2008.08.076. Epub 2008 Aug 26.

Abstract

WNK kinases have rapidly emerged as important regulators of Na+ and K+ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K+ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordon's syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (DeltaAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, DeltaAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Cell Membrane
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Oocytes / metabolism
  • Peptides / genetics
  • Peptides / metabolism
  • Potassium Channels, Inwardly Rectifying / genetics
  • Potassium Channels, Inwardly Rectifying / metabolism*
  • Protein Interaction Mapping
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Xenopus laevis

Substances

  • KCNJ1 protein, human
  • Peptides
  • Potassium Channels, Inwardly Rectifying
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Protein-Serine-Threonine Kinases
  • WNK4 protein, human