Endoplasmic reticulum–associated degradation of the renal potassium channel, ROMK, leads to type II Bartter syndrome
Journal of Biological Chemistry
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 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 steadystate 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. http://www.jbc.org/cgi/