Endoplasmic reticulum-associated degradation of the renal potassium channel, ROMK, leads to type II Bartter syndrome

J Biol Chem. 2017 Aug 4;292(31):12813-12827. doi: 10.1074/jbc.M117.786376. Epub 2017 Jun 19.


Type II Bartter syndrome is caused by mutations in the renal outer medullary potassium (ROMK) channel, but the molecular mechanisms underlying this disease are poorly defined. To rapidly screen for ROMK function, we developed a yeast expression system and discovered that yeast cells lacking endogenous potassium channels could be rescued by WT ROMK but not by ROMK proteins containing any one of four Bartter mutations. We also found that the mutant proteins were significantly less stable than WT ROMK. However, their degradation was slowed in the presence of a proteasome inhibitor or when yeast cells contained mutations in the CDC48 or SSA1 gene, which is required for endoplasmic reticulum (ER)-associated degradation (ERAD). Consistent with these data, sucrose gradient centrifugation and indirect immunofluorescence microscopy indicated that most ROMK protein was ER-localized. To translate these findings to a more relevant cell type, we measured the stabilities of WT ROMK and the ROMK Bartter mutants in HEK293 cells. As in yeast, the Bartter mutant proteins were less stable than the WT protein, and their degradation was slowed in the presence of a proteasome inhibitor. Finally, we discovered that low-temperature incubation increased the steady-state levels of a Bartter mutant, suggesting that the disease-causing mutation traps the protein in a folding-deficient conformation. These findings indicate that the underlying pathology for at least a subset of patients with type II Bartter syndrome is linked to the ERAD pathway and that future therapeutic strategies should focus on correcting deficiencies in ROMK folding.

Keywords: 70-kilodalton heat shock protein (Hsp70); Cdc48; ER quality control; Saccharomyces cerevisiae; endoplasmic reticulum–associated protein degradation (ERAD); proteasome; yeast.

MeSH terms

  • Adenosine Triphosphatases / antagonists & inhibitors
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Substitution
  • Animals
  • Bartter Syndrome / genetics*
  • Bartter Syndrome / metabolism
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Endoplasmic Reticulum-Associated Degradation* / drug effects
  • Enzyme Inhibitors / pharmacology
  • HEK293 Cells
  • HSP70 Heat-Shock Proteins / chemistry
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism
  • Hot Temperature
  • Humans
  • Microbial Viability
  • Models, Molecular*
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Point Mutation*
  • Potassium Channels, Inwardly Rectifying / chemistry
  • Potassium Channels, Inwardly Rectifying / genetics*
  • Potassium Channels, Inwardly Rectifying / metabolism
  • Proteasome Inhibitors / pharmacology
  • Protein Interaction Domains and Motifs
  • Protein Stability / drug effects
  • Proteolysis / drug effects
  • Rats
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Valosin Containing Protein


  • Cell Cycle Proteins
  • Enzyme Inhibitors
  • HSP70 Heat-Shock Proteins
  • KCNJ1 protein, human
  • Kcnj1 protein, rat
  • Mutant Proteins
  • Potassium Channels, Inwardly Rectifying
  • Proteasome Inhibitors
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphatases
  • SSA1 protein, S cerevisiae
  • CDC48 protein, S cerevisiae
  • Valosin Containing Protein

Supplementary concepts

  • Bartter syndrome, antenatal , type 2