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 7

ARH couples YxNPxFV signal recognition to clathrin coated-pit localization.

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ARH couples YxNPxFV signal recognition to clathrin coated-pit localizati...
(A) Cartoon of the ARH domain structure. WT ARH is compared with ARH1–187, a truncation mutant comprised of the PTB domain. (B) Interaction of ARH with ROMK, clathrin, and AP-2 was assessed by anti myc-immunoprecipitation. COS-7 cells were cotransfected with HA-tagged ROMK and either myc-tagged WT ARH (left columns) or ARH1–187 (right columns). Myc-antibody (+) or mock antibody (-) immunoprecipitation was followed by immunoblotting for ROMK (HA antibody), clathrin (anti–heavy chain antibody), or AP-2 (α-adaptin antibody) and compared with input. (C) ROMK endocytosis measured by biotinylation in cells cotransfected with WT ARH, ARH1–187, or empty vector (-). Surface biotin-labeled ROMK internalized after a 5-minute chase is compared with the total surface pool. (D) Quantification of ROMK endocytosis of groups above. (n = 3; *P < 0.001 greater than control; #P < 0.05 less than control.) (E) siRNA-mediated knockout of endogenous ARH blocks ROMK endocytosis in COS cells. ARH protein and ROMK endocytosis was measured in cells cotransfected with ROMK or no siRNA (MOCK), nontargeting siRNA (control [-cont]) or ARH-specific siRNA probes. The top 2 panels are representative immunoblots of ARH relative to a loading control (COX IV). The bottom 2 panels are surface-labeled ROMK internalized after a 5-minute chase compared with the total surface pool. (F) Quantification of ROMK endocytosis in groups above. n = 3; #P < 0.05 less than both controls.