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The Rab‐GAP, AS160, participates in the regulation of apical membrane epithelial sodium channel (ENaC) density and recycling
Author(s) -
Liang Xiubin,
Butterworth Michael B.,
Peters Kathryn W.,
Frizzell Raymond A.
Publication year - 2010
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.24.1_supplement.1024.1
Subject(s) - epithelial sodium channel , phosphorylation , microbiology and biotechnology , rab , chemistry , gene knockdown , biology , sodium , biochemistry , gtpase , apoptosis , organic chemistry
The Rab‐GAP, AS160 is a recently discovered Akt kinase substrate. Since phosphorylation sites for Akt are similar to those for the aldosterone (Aldo)‐induced kinase, SGK1, we postulated that phosphorylation of AS160 may promote ENaC trafficking during aldo stimulation. AS160 expression was detected in mCCD cells, in which its expression was increased by its transfection or reduced by shRNA‐mediated knockdown, followed by assays of ENaC expression and function. Aldo induced a time‐dependent increase in AS160 expression, and its phosphorylation at five sites: T568, S588, T642, S318 and S751, detected by phospho‐specific antibodies. Under non‐stimulated (basal) conditions, AS160 knockdown increased apical ENaC without changing total ENaC expression and increased Na transport. After AS160 knockdown, aldo did not further augment apical ENaC density or Na transport. Under basal conditions, AS160 over‐expression increased total ENaC expression 2.5‐fold but did not increase surface ENaC expression. However, aldo increased surface ENaC expression and Na current 2.5‐fold in epithelia expressing AS160. These findings suggest that AS160 normally retains ENaC within intracellular compartments, and that SGK1‐mediated AS160 phosphorylation blocks its Rab‐GAP activity, permitting ENaC trafficking to the apical membrane in response to aldosterone. [Supported by AHA fellowship 0725416U and NIH DK54814]