Phosphatidylinositol 3,4,5-trisphosphate mediates aldosterone stimulation of epithelial sodium channel (ENaC) and interacts with gamma-ENaC.
Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). PIP(3) significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP(3)-binding protein (Grp1-PH), which sequestered intracellular PIP(3). 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(T)-ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).
Helms
My N
MN
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Liu
Lian
L
Liang
You-You
YY
Al-Khalili
Otor
O
Vandewalle
Alain
A
Saxena
Sunil
S
Eaton
Douglas C
DC
Ma
He-Ping
HP
D000450:Aldosterone / Q000494:pharmacology*; D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004347:Drug Interactions; D004553:Electric Conductivity; D053503:Epithelial Sodium Channels; D015870:Gene Expression; D007668:Kidney; D051379:Mice; D018613:Microscopy, Confocal; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection
D053503:Epithelial Sodium Channels; D010446:Peptide Fragments; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; C060974:phosphatidylinositol 3,4,5-triphosphate; D000450:Aldosterone; D000584:Amiloride