WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells

Kidney Int. 2006 Jun;69(12):2162-70. doi: 10.1038/sj.ki.5000333. Epub 2006 May 10.


Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotinylation
  • Blotting, Western
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Dynamin II / pharmacology
  • Dynamins / pharmacology
  • Epithelial Cells / chemistry
  • Epithelial Cells / cytology
  • Epithelial Cells / physiology*
  • Fluorescent Antibody Technique
  • Gene Expression Profiling
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / physiology
  • Humans
  • Immunoprecipitation
  • Kidney / chemistry
  • Kidney / cytology
  • Kidney / physiology*
  • Lysosomes / drug effects
  • Lysosomes / physiology
  • Mutation
  • Protein Structure, Tertiary / physiology
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / pharmacology
  • Protein-Serine-Threonine Kinases / physiology*
  • Pseudohypoaldosteronism / etiology
  • Pseudohypoaldosteronism / genetics*
  • Pseudohypoaldosteronism / physiopathology
  • Sodium Chloride Symporters / drug effects*
  • Sodium Chloride Symporters / genetics
  • Sodium Chloride Symporters / physiology*


  • Sodium Chloride Symporters
  • Protein-Serine-Threonine Kinases
  • WNK4 protein, human
  • Dynamin II
  • Dynamins