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The Role of Endoplasmic Reticulum–Associated Degradation in the Regulation of a Potassium Channel Associated with Type II Bartter Syndrome
Author(s) -
O'Donnell Brighid,
Mackie Timothy,
Brodsky Jeffrey
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb182
Subject(s) - bartter syndrome , mutant , protein degradation , biology , endoplasmic reticulum associated protein degradation , medicine , chemistry , endocrinology , microbiology and biotechnology , endoplasmic reticulum , unfolded protein response , biochemistry , gene , hypokalemia
Type II Bartter Syndrome is an autosomal recessive disease caused by a loss of function mutation in the human KCNJ1 gene. This gene encodes the Renal Outer Medullary Potassium channel (ROMK), the primary route for K + efflux within the nephron. To maintain K + homeostasis, renal epithelial cells must regulate the apical surface density of ROMK. The molecular defects in ROMK channel trafficking and regulation that cause Bartter Syndrome are poorly defined, but we propose that some of the disease‐causing mutations lead to protein misfolding. Because misfolded membrane proteins are subject to ER quality control, we asked if some mutations target ROMK for Endoplasmic Reticulum–Associated Degradation (ERAD) in a yeast model. The ability of wild‐type and Bartter ROMK mutants to rescue low K + sensitivity was investigated. Yeast lacking Trk1 and Trk2 K + channel proteins were transformed with mutant ROMK, serially diluted, and plated onto media of various K + concentrations. Growth was scored via imaging, and relative ROMK expression levels were assayed by Western blot. Wild type ROMK and Bartter mutant stability was analyzed by cycloheximide chase analysis. To half of each culture, MG132 was added to inhibit the proteosome, and protein translation was halted with cycloheximide. Aliquots were collected at various time points, and relative levels of ROMK were assessed by Western blot. We found that yeast expressing ROMK Bartter mutants exhibit growth defects on low K + media, and the mutant proteins are less stable compared to wild‐type ROMK. Degradation is proteasome‐mediated. Bartter Syndrome mutants increase targeting for ERAD when compared to wild‐type ROMK. NIH Grant 5T32DK071492‐09 and R01GM075061