The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney
Liang Fang, … , James B. Wade, Paul A. Welling
Liang Fang, … , James B. Wade, Paul A. Welling
Published October 19, 2009
Citation Information: J Clin Invest. 2009;119(11):3278-3289. https://doi.org/10.1172/JCI37950.
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Research Article Nephrology

The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney

Abstract

Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical “NPXY” endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis.

Authors

Liang Fang, Rita Garuti, Bo-Young Kim, James B. Wade, Paul A. Welling

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Figure 2

Colocalization and coimmunoprecipitation of ARH and ROMK in the rat kidney.

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Colocalization and coimmunoprecipitation of ARH and ROMK in the rat kidn...
Immunofluorescent staining of (A) ROMK with a chicken anti-ROMK antibody and (B) ARH with a goat anti-ARH antibody (C) and colocalization of ARH (green) and ROMK (red) in a rat kidney collecting duct, using appropriate, specific Alexa Fluor–conjugated secondary antibodies. Arrowheads indicate subapical or perinuclear labeling in principal cells; asterisks indicate intercalated cells. Scale bar: 8.5 μm. (D) Low-power confocal microscope image of ARH localization in cortex of rat kidney. ARH can be detected in cortical collecting ducts (CCDs), TALs, and distal tubules (DTs), but little or no labeling is detected in proximal tubules (asterisks). Collecting ducts were identified based on double labeling with antibody to AQP2 raised in chicken and other segments identified based on morphological criteria. Scale bar: 8 μm. (E) Immunoprecipitation analysis of ROMK channel with ARH from whole rat kidney extracts. (F) Immunoprecipitation of HA epitope–tagged ROMK or mutant ROMK, lacking the NPNF motif, with myc-tagged ARH from COS-7 cells transfected with indicated (+) cDNAs. Only WT ROMK was immunoprecipitated with ARH. (G) Quantification of IP results. n = 3; *P < 0.001.