Endoplasmic Reticulum Associated Degradation (ERAD) of the Renal Outer Medullary Potassium Channel, ROMK, Underlies Type II Bartter Syndrome

Type II Bartter Syndrome is an autosomal recessive disease caused by mutations in the human KCNJ1 gene, which encodes the Renal Outer Medullary Potassium channel (ROMK). To maintain potassium homeostasis, renal epithelial cells regulate the apical surface density of ROMK. The molecular defects in ROMK channel trafficking and regulation that cause Bartter Syndrome are poorly defined. We propose that some of the disease‐causing mutations lead to protein misfolding. Because misfolded membrane proteins are subject to ER quality control, we examined if select mutations target ROMK for En‐doplasmic Reticulum Associated Degradation (ERAD) in a new yeast model. Four Bartter mutations within the immunoglobulin‐like domain in ROMK were selected due to their close proximity to regions rich in β‐pleated sheets. ROMK expression vectors encoding the wild type and mutant proteins were transformed into strains lacking PDR5 , which mediates drug efflux. The transformed yeast were treated with MG132 (a proteasome inhibitor) and compared with those treated with DMSO as a control. Next, because all ubiquitinated ERAD substrates are extracted from the ER by the Cdc48 (in yeast) or the homologous p97 (in mammals) complex, we transformed ROMK into a temperature sensitive yeast strain, cdc48‐2, and assessed the rate of degradation. Finally, because ERAD substrates are selected by molecular chaperones prior to ubiquitination and proteasome targeting, we then measured degradation in SSA1 yeast and ssa1‐45 yeast, which encodes a temperature‐sensitive mutation in the primary Hsp70. Consistent with our hypothesis, we discovered that inhibition of the proteasome stabilized the Bartter mutants, and that wild‐type ROMK was significantly more stable than any of the Bartter mutants examined. Both Cdc48 and Hsp70 were also required for Bartter mutant degradation. In addition, the growth of yeast lacking endogenous potassium channels could be rescued by wild type ROMK but not by the mutant proteins. We then used sucrose gradient centrifugation and indirect immunofluorescence microscopy to determine channel residence, and both studies indicated that the majority of ROMK was ER localized, consistent with it being subjected to ERAD. Last, we performed cycloheximide chase reactions in the presence or absence of MG132 in a HEK293 cell line transiently expressing either ROMK or each of the Bartter mutants. The data mirrored those obtained in yeast: inhibiting the proteasome generated more significant stabilization of the Bartter mutants, and the relative instability of the mutants was greater than the wild type protein. These data indicate that specific Bartter mutations in the immunoblobulin‐like domain target ROMK for ERAD, thus providing a molecular basis for at least some forms of the disease. In the future, we hope to identify drug targets for patients with these mutations by using the yeast and mammalian expression systems. Support or Funding Information This work was funded by National Institute of Health grants GM75061 to J.L.B. and DK79307 (University of Pittsburgh George O'Brien Kidney Research Center), and by NIH Grants 5T32DK071492‐09, 5T32DK071492‐10, and 4K12HD052892‐10.