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Ankyrin G Alters ENaC Membrane Delivery to Increase Na + Transport in the Distal Kidney Nephron
Author(s) -
Klemens Christine Anne,
Kightlinger Lindsay,
Liu Xiaoning,
Edinger Robert,
Butterworth Michael Bruce
Publication year - 2016
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.30.1_supplement.1223.2
Subject(s) - epithelial sodium channel , reabsorption , renal sodium reabsorption , apical membrane , medicine , endocrinology , aldosterone , nephron , biology , microbiology and biotechnology , sgk1 , ankyrin , chemistry , kidney , sodium , biochemistry , phosphorylation , membrane , organic chemistry , gene
The heterotrimeric epithelial sodium channel (ENaC) comprised of α, β, and γ subunits, is the predominant aldosterone regulated sodium (Na + ) channel in the kidney distal nephron, and plays an essential role in fine‐tuning Na + reabsorption into the blood. Stimulation by aldosterone induces a number of changes to increase urinary Na + reabsorption, including increasing ENaC subunit translation and modifying key regulators that alter ENaC function and/or membrane density. We identified ankyrin G (AnkG) to be a novel, aldosterone stimulated, ENaC regulator. Knock down of AnkG expression reduces the endogenous amiloride sensitive (ENaC) current by 69%±1.57% (n=15, p<0.0001), while over expression of AnkG increases ENaC current by 67%±11% (n=12, p=0.0001) in mouse cortical collecting duct (mCCD) cells. Membrane permeabilization verified that this alteration of Na + transport was through ENaC and not a secondary effect of intracellular driving force via altered Na + K + ATPase. The aim of this study was to determine the mechanism behind AnkG's regulation of ENaC activity, whether through increased surface delivery, altered channel open probability, increased membrane dwell time, increased intracellular recycling, or blunted internalization and degradation. We propose that AnkG expression modulates Na + reabsorption in collecting duct cells by increasing ENaC delivery to the surface, thereby increasing the number of channels present at the apical membrane. Biotinylation of surface βENaC determined that AnkG over expression increases the number of channels at the surface 71%±26% (n=4, p‐value=0.02). Using a combination of electrical and biochemical pulse‐chase assays, we found that AnkG expression does not alter ENaC internalization rates or ENaC recycling; however, mCCD cells with reduced AnkG had significantly slower apical delivery rates (k=0.1361 min −1 ±0.0081vs k=0.1867 min −1 ±0.0045, n=12, p<0.0001), while AnkG over expression in mCCDs accelerated ENaC delivery (k=0.1724 min −1 ±.0020vs k=0.1298 min −1 ±.0035, n=9, p<0.0001). Neither AnkG knock down or over expression altered the total amount of ENaC protein in the mCCD cells, suggesting AnkG is impacting ENaC delivery rates, rather than the quantity of ENaC delivered to the surface. These findings highlight the role of AnkG as an aldosterone induced protein and novel regulator of ENaC that could alter Na + reabsorption in the distal nephron. Ongoing studies aim to fully characterize the molecular mechanisms underlying this dynamic interaction. Support or Funding Information 15PRE25680068, DK079307, UPMC Cell Biology Teaching Fellowship