TY - JOUR
T1 - The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK)
AU - Mackie, Timothy D.
AU - Kim, Bo Young
AU - Subramanya, Arohan R.
AU - Bain, Daniel J.
AU - O'Donnell, Allyson F.
AU - Welling, Paul A.
AU - Brodsky, Jeffrey L.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants GM75061 and DK79307 (University of Pittsburgh George O’Brien Kidney Research Center) (to J. L. B.), R01DK098145 (to A. R. S.), and DK054231 (to P. A. W.) and by National Science Foundation MCB CAREER Grant 1553143 (to A. F. O.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported by National Institutes of Health Grants GM75061 and DK79307 (University of Pittsburgh George O'Brien Kidney Research Center) (to J. L. B.), R01DK098145 (to A. R. S.), and DK054231 (to P. A. W.) and by National Science Foundation MCB CAREER Grant 1553143 (to F. O.).
PY - 2018/3/2
Y1 - 2018/3/2
N2 - Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the renal outer medullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast Saccharomyces cerevisiae. We determined that ROMK primarily resides in the endoplasmic reticulum (ER), as it does in mammalian cells, and is subject to ER-associated degradation (ERAD). However, sufficient ROMK levels on the plasma membrane rescued growth on low-potassium medium of yeast cells lacking endogenous potassium channels. Next, we aimed to identify the biological pathways most important forROMKregulation. Therefore, we used a synthetic genetic array to identify non-essential genes that reduce the plasma membrane pool of ROMK in potassium-sensitive yeast cells. Genes identified in this screen included several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Mass spectroscopy analysis confirmed that yeast cells lacking an ESCRT component accumulate higher potassium concentrations. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in HEK293 cells. Our results indicate that components of the post-endocytic pathway influence the cell-surface density of ROMKand establish that components in this pathway modulate channel activity.
AB - Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the renal outer medullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast Saccharomyces cerevisiae. We determined that ROMK primarily resides in the endoplasmic reticulum (ER), as it does in mammalian cells, and is subject to ER-associated degradation (ERAD). However, sufficient ROMK levels on the plasma membrane rescued growth on low-potassium medium of yeast cells lacking endogenous potassium channels. Next, we aimed to identify the biological pathways most important forROMKregulation. Therefore, we used a synthetic genetic array to identify non-essential genes that reduce the plasma membrane pool of ROMK in potassium-sensitive yeast cells. Genes identified in this screen included several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Mass spectroscopy analysis confirmed that yeast cells lacking an ESCRT component accumulate higher potassium concentrations. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in HEK293 cells. Our results indicate that components of the post-endocytic pathway influence the cell-surface density of ROMKand establish that components in this pathway modulate channel activity.
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U2 - 10.1074/jbc.M117.819086
DO - 10.1074/jbc.M117.819086
M3 - Article
C2 - 29311259
AN - SCOPUS:85042937682
SN - 0021-9258
VL - 293
SP - 3201
EP - 3217
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 9
ER -