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A Screen for Potassium‐sensitivity in Yeast Reveals Endosomal Sorting Factors that Regulate the Renal Outer Medullary Potassium (ROMK) Channel
Author(s) -
Mackie Timothy D.,
Welling Paul A.,
Subramanya Arohan R.,
Brodsky Jeffrey L.
Publication year - 2017
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.31.1_supplement.703.18
Subject(s) - endosome , endocytic cycle , reabsorption , microbiology and biotechnology , distal convoluted tubule , potassium channel , chemistry , bartter syndrome , biology , medicine , endocrinology , endocytosis , biochemistry , kidney , hypokalemia , cell , intracellular
The renal outer medullary potassium (ROMK) channel is a major route for potassium secretion from distal nephron principal cells in response to changes in dietary potassium intake. In addition, ROMK acts in tandem with the sodium‐potassium‐chloride cotransporter‐2 (NKCC2) to facilitate sodium reabsorption in the thick ascending limb. Genetic perturbations in ROMK cause an autosomal recessive salt‐wasting disorder in newborns known as Bartter Syndrome Type II, while these same mutations provide heterozygote individuals with resistance to developing hypertension. To date, ROMK anterograde trafficking through the secretory pathway and endocytosis from the plasma membrane in renal epithelia are well studied, however little is known about the factors that regulate either the post‐endocytic fate of ROMK or the fate of ROMK in the early secretory pathway. To rectify this deficiency in our understanding of how ROMK is regulated, we developed an assay utilizing a genetic screening technique in the budding yeast Saccharomyces cerevisiae . In brief, yeast were rendered sensitive to low potassium by deletion of the two native potassium transporters, Trk1 and Trk2. ROMK was then transformed into these cells, and crossed with a collection of 5,000 unique strains, each of which have a single ORF deleted. Strains that grow better on low potassium media are hypothesized to be deficient in negative regulators of ROMK. Several factors related to endosomal sorting were identified through this screen, including members of the class‐C core vacuole‐endosome tethering (CORVET) complex, which brings endocytic vesicles together to form early endosomes, as well as the endosomal complexes required for transport (ESCRT), which sort ubiquitinated cargo into multi‐vesicular bodies prior to degradation in the lysosome/vacuole. These findings were validated by serial dilution growth assays in independently constructed yeast strains. Next, these findings were validated in a more relevant model. Cell surface biotinylation of HEK293 cells treated with siRNA to knockdown human orthologues of screen hits indicated that CORVET and ESCRT also deplete ROMK from the plasma membrane in human cells. These genetic data lead us to propose that the plasma membrane population of ROMK is endocytosed and sorted to the lysosome for degradation in a CORVET and ESCRT‐dependent manner. In addition, hypothesis‐driven studies in yeast showed that ROMK is also partially regulated at the level of the endoplasmic reticulum (ER) via ER‐associated degradation (ERAD). Together, these findings enhance our understanding of the cellular itinerary of this critical ion channel and may lead to the development of novel therapies for patients with Bartter syndrome and other disorders of potassium balance. Support or Funding Information This work was supported by grants GM75061 and DK79307 to J.L.B from the National Institutes of Health