Amiloride-sensitive epithelial Na؉ channels (ENaC) are responsible for trans-epithelial Na

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits α, β, and γ in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 ± 1.1 pS and an Na+-to-K+selectivity of 0.97 ± 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (α, β, and γ) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the α-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the β- and γ-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the α-subunit of ENaC alone or in combination with some protein other than the β- or γ-subunit protein is the major component of lung alveolar epithelial cation channels.

Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the β- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP(2). Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.

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...

Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.