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Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane
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WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells

H Cai1, V Cebotaru, Y-H Wang

  • 1Division of Nephrology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Kidney International
|May 12, 2006
PubMed

Insights

Pseudohypoaldosteronism type II (PHA II) involves WNK kinase mutations. Wild-type WNK4 inhibits sodium chloride cotransporter (NCC) surface expression, likely via lysosomal degradation, not endocytosis.

Area of Science:

  • Molecular biology
  • Cell biology
  • Physiology

Background:

  • Pseudohypoaldosteronism type II (PHA II) is linked to mutations in WNK (with no lysine (k)) kinases.
  • Wild-type WNK4 (WNK4 WT) is known to inhibit the activity and surface expression of the sodium chloride cotransporter (NCC).

Purpose of the Study:

  • To investigate the impact of WNK4 WT and its mutants (E562K, R1185C) on NCC protein processing in mammalian cells.
  • To elucidate the mechanism by which WNK4 affects NCC surface expression.

Main Methods:

  • Surface biotinylation assays to quantify cell surface NCC.
  • Western blotting to assess protein levels.
  • Co-immunoprecipitation (Co-IP) to study protein interactions.
  • Immunostaining for cellular localization.
  • Treatment with dynamin inhibitors and a proton pump inhibitor (bafilomycin A1).

Main Results:

  • WNK4 WT significantly reduced NCC surface expression (58.9%) in Cos-7 cells.
  • The WNK4 mutant E562K showed no significant effect on NCC surface expression (92.9%).
  • The WNK4 mutant R1185C partially reduced NCC surface expression (76.2%).
  • WNK4 WT's effect was independent of dynamin and clathrin-mediated endocytosis.
  • WNK4 WT and WNK4 E562K interacted with NCC.
  • Bafilomycin A1 partially reversed WNK4 WT's inhibitory effect on NCC expression.

Conclusions:

  • WNK4 WT significantly inhibits NCC surface expression.
  • This inhibition is likely mediated by enhanced lysosomal degradation of NCC, rather than increased clathrin-mediated endocytosis.
  • WNK4 kinase mutations in PHA II may disrupt normal NCC regulation through altered protein processing and degradation pathways.

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