Phosphorylation‐Dependent Endocytosis of ROMK Limits Urinary Potassium Loss

ROMK channels in the aldosterone‐sensitive distal nephron are tightly regulated to maintain potassium balance. In response to potassium restriction, ROMK channels are internalized from the apical membrane, safeguarding against extensive potassium loss. Previously, we found the clathrin adaptor protein, ARH, directly binds to ROMK to stimulate endocytosis (Fang et al, JCI ‘09). WNK signaling pathway has been implicated in activating ROMK endocytosis, but the mechanism is not clear, and physiological significance of ARH‐mediated ROMK regulation remains uncertain. To tackle these questions, we explored how WNK kinases activate ARH and compared the homeostatic response to dietary potassium restriction in wild type and ARH KO mice. In vitro phosphorylation studies revealed ARH is phosphorylated by ERK, which in turn is activated by L‐WNK1 or WNK4. Examination of the phosphorylation cascade revealed that dietary potassium restriction increases phosphorylation of ERK and ARH‐S14 in the kidney, coincident with phospho‐activation of the WNKs, and reduction of ROMK apical surface expression. We found phosphorylation of ARH at S14 prevents its degradation, stimulates its membrane association, and this enhances ROMK endocytosis. ARH KO mice exhibit an increase in glycosylated ROMK in the renal cortex without changes in the abundance of the core‐glycosylated or unglycosylated forms, consistent with inhibition of post‐endocytic degradation. In dietary potassium deficiency, ARH KO mice are unable to reduce ROMK protein to the same extent as WT mice, and thus exhibit excessive urinary potassium loss compared to WT mice. These studies identify ARH as a downstream effector of the WNK/ERK pathway that stimulates ROMK endocytosis in potassium deficiency.