Phosphatidylinositol 3,4,5-Trisphosphate Mediates Aldosterone Stimulation of Epithelial Sodium Channel (ENaC) and Interacts with γ-ENaC

Abstract

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 ␥-epithelial sodium channel (␥-ENaC) were truncated by PCRbased mutagenesis (␥ T -ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing ␣-and ␤-ENaC only or with either ␥-ENaC or ␥ 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 plasma membrane. These data suggest that PIP 3 mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of ␥-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between ␥-ENaC and PIP 3 . ENaC2 is a member of the ENaC/Deg superfamily of ion channels responsible for sodium transport across the apical membrane of a variety of epithelia including the colon, lung, and kidney (reviewed in Ref. 1). Since 1994, when ENaC was initially cloned from rat colon (2), the biophysical properties and molecular structure of ENaC have been extensively studied. Several lines of evidence suggest that ENaC is composed of three subunits, ␣, ␤, and ␥, and that all three subunits are required to form a functional ␣␤␥-ENaC channel complex (2-9).Studying the mechanisms that regulate ENaC function is important because abnormal channel activity leads to several severe diseases. Constitutive activation of any component of ENaC subunits can cause Liddle's syndrome, an autosomal dominant inherited disease that causes excessive sodium retention and hypertension. Conversely, loss of function mutations in ␣-, ␤-, or ␥-ENaC causes pseudohypoaldosteronism type I, a hypotensive condition characterized by an inability to retain salt. These syndromes highlight the importance of normal ENaC activity in the kidney to maintain fluid and sodium homeostasis. The proper regulation of ENaC activity is also very important in the lung, because transgenic mice lacking functional channels die within 40 h of birth from fluid filled airways (10). Additionally, increases in intracellular Cl Ϫ concentrations that secondarily lead to changes in ENaC activity play an important role in the pathophysiology of cystic fibrosis (11).Anionic phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), are normally located in the inner leaflet of the plas...

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells

“…Although it is clear that glucocorticoid hormones do induce SGK1 expression (31), SGK1 is an important downstream target of PI3K (29), and data from several absorptive cell types have suggested that insulin can increase the apical abundance of ENaC via a mechanism dependent on a PI3K-mediated increase in SGK1 activity (2,8,46,51). Moreover, PIP 2 and PIP 3 also activate ENaC by binding to the channel complex, and this provides another mechanism by which PI3K-coupled agonists, such as insulin, can stimulate Na ϩ transport (20,42,43,52). However, although the present data show that insulin stimulation/PI3K-P110␣ expression can control G Cl , these maneuvers had no effect on G Na in hormonedeprived cells but augmented the dexamethasone/SGK1-induced G Na .…”

“…These anionic phospholipids control many aspects of cell physiology by activating PI3K and downstream kinases such as SGK1 (15,29) and by directly controlling ion channel activity (see, for example, Refs. 20,22). Since PI3K has also been reported to control G Cl in arterial smooth muscle (13), subsequent experiments explored the effects of artificially increasing cellular PI3K activity by transiently expressing membraneanchored PI3K-P110␣ (see Ref.…”

“…Moreover, insulin clearly regulates Na ϩ transport in the aldosterone-sensitive epithelia of the distal nephron, and this seems to involve control over the apical abundance and/or activity of the epithelial Na ϩ channel (ENaC) (see, for example, Refs. 7,20), a transport protein composed of three homologous subunits (␣-, ␤-, and ␥-ENaC), which appears to allow apical Na ϩ entry in absorptive epithelia (25). The initial aim of the present study was therefore to determine the extent to which insulin can exert such control over Na ϩ transport in pulmonary epithelia, and we have therefore 1) recorded membrane currents from a well-characterized, Na ϩ -absorbing cell line derived from the human distal airway epithelium (H441; see, for example, Refs.…”

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

“…42 Briefly, the mpkCCD c14 cells were incubated in a 1:1 mix of DMEM:F12 medium (Invitrogen, Carlsbad, CA) supplemented with 50 nM dexamethasone, 1 nM triiodothyronine, 20 mM Hepes, 2 mM L-glutamine, 0.1% penicillin/ streptomycin, and 2% heat-inactivated FBS. Cells were maintained at 37°C with 5% CO 2 in air for 2 weeks until they formed a confluent monolayer and became fully polarized.…”

Acute Regulation of the Epithelial Na+ Channel by Phosphatidylinositide 3-OH Kinase Signaling in Native Collecting Duct Principal Cells

“…Phosphatidylinositide 3-OH kinase (PI3-K) is central to many cellular signaling pathways that control ENaC activity (8 -10). In particular, PI3-K is an effector that is necessary for aldosterone and insulin control of the channel (11)(12)(13)(14)(15). Both aldosterone and insulin stimulate PI3-K signaling and increase the levels of the phosphoinositide products of this kinase in renal epithelial cells (13,16).…”