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Molecular mechanism of ROMK channel endocytosis
Author(s) -
Fang Liang,
Kim Boyoung,
Wade James B.,
Welling Paul A.
Publication year - 2008
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.22.1_supplement.1158.9
Subject(s) - internalization , endocytosis , microbiology and biotechnology , chemistry , clathrin , potassium channel , dynamin , biophysics , biochemistry , biology , cell
The apical surface density of ROMK channels in the renal CCD is down regulated by clathrin‐dependent endocytosis upon dietary potassium restriction. Aberrant stimulation of ROMK endocytosis may contribute to hyperkalemia in pseudohypoaldosteronism type II. ROMK contains an “NPXY‐type” internalization motif but the endocytotic machinery that recognizes the signal has been a mystery. Here we show that a member of a new class of clathrin adaptors, ARH, controls this activity. We found ARH is predominantly expressed in the distal nephron, where it co‐localizes with ROMK in subapical and perinuclear vesicles. As measured by cell surface biotinylation or antibody binding, ARH expression led to a dramatic attenuation of ROMK surface expression. A close relative of ROMK, Kir2.1, does not contain an “NPXY” type signal and is not affected by ARH. Swapping the C‐terminal domain of ROMK, containing the NPXF motif, onto the Kir2.1 channel conferred ARH‐dependent internalization. Alanine replacement of the NPXF motif increased cell surface expression and rendered the chimeric channel resistant to ARH. A “pull‐down” approach with recombinant ARH and GST‐ROMKC proteins revealed that ARH directly interacts with the ROMK C‐terminus in a NPNF signal dependent manner. In conclusion, ARH marks ROMK channels for endocytosis through direct interaction with the NPXF internalization signal. NIH RO1 DK63049 and DK54231.