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

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