Otor Al-Khalili

Antisense oligonucleotides against the α-subunit of ENaC decrease lung epithelial cation-channel activity

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.

Mechanisms of aldosterone's action on epithelial Na + transport.

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.

Phosphatidylinositol 3,4,5-Trisphosphate Mediates Aldosterone Stimulation of Epithelial Sodium Channel (ENaC) and Interacts with γ-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 (PIP3). PIP3 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 PIP3-binding protein (Grp1-PH), which sequestered intracellular PIP3. 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of γ-epithelial sodium channel (γ-ENaC) were truncated by PCRbased mutagenesis (γT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing α- and β-ENaC only or with either γ-ENaC or γT-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 PIP3 mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of γ-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between γ-ENaC and PIP3.

S-Adenosyl-l-homocysteine Hydrolase Regulates Aldosterone-induced Na+ Transport

Aldosterone-induced Na+reabsorption, in part, is regulated by a critical methyl esterification; however, the signal transduction pathway regulating this methylation remains unclear. The A6 cell line was used as a model epithelia to investigate regulation of aldosterone-induced Na+ transport byS-adenosyl-l-homocysteine hydrolase (SAHHase), the only enzyme in vertebrates known to catabolizeS-adenosyl-l-homocysteine (SAH), an end product inhibitor of methyl esterification. Sodium reabsorption was decreased within 2 h by 3-deazaadenosine, a competitive inhibitor of SAHHase, with a half inhibitory concentration between 40 and 50 μm. Aldosterone increased SAH catabolism by activating SAHHase. Increased SAH catabolism was associated with a concomitant increase in S-adenosylmethionine catabolism. Moreover, SAH decreased substrate methylation. Antisense oligonucleotide complementary to SAHHase mRNA decreased SAHHase activity and Na+ current by approximately 50%. Overexpression of SAHHase increased SAHHase activity and dependent substrate methyl esterification. Whereas basal Na+ current was not affected by overexpression of SAHHase, aldosterone-induced current in SAHHase-overexpressing cells was significantly potentiated. These results demonstrate that aldosterone induction of SAHHase activity is necessary for a concomitant relief of the methylation reaction from end product inhibition by SAH and the subsequent increase in Na+ reabsorption. Thus, regulation of SAHHase activity is a control point for aldosterone signal transduction, but SAHHase is not an aldosterone-induced protein.

Isoprenylcysteine-O-carboxyl methyltransferase regulates aldosterone- sensitive Na+ reabsorption

The Xenopus laevis distal tubule epithelial cell line A6 was used as a model epithelia to study the role of isoprenylcysteine-O-carboxyl methyltransferase (pcMTase) in aldosterone-mediated stimulation of Na+transport. Polyclonal antibodies raised against X. laevispcMTase were immunoreactive with a 33-kDa protein in whole cell lysate. These antibodies were also reactive with a 33-kDa product from in vitro translation of the pcMTase cDNA. Aldosterone application increased pcMTase activity resulting in elevation of total protein methyl esterification in vivo, but pcMTase protein levels were not affected by steroid, suggesting that aldosterone increased activity independent of enzyme number. Inhibition of pcMTase resulted in a reduction of aldosterone-induced Na+transport demonstrating the necessity of pcMTase-mediated transmethylation for steroid induced Na+ reabsorption. Transfection with an eukaryotic expression construct containing pcMTase cDNA increased pcMTase protein level and activity. This resulted in potentiation of the natriferic actions of aldosterone. However, overexpression did not change Na+ reabsorption in the absence of steroid, suggesting that pcMTase activity is not limiting Na+ transport in the absence of steroid, but that subsequent to aldosterone addition, pcMTase activity becomes limiting. These results suggest that a critical transmethylation is necessary for aldosterone-induction of Na+ transport. It is likely that the protein catalyzing this methylation is isoprenylcysteine-O-carboxyl methyltransferase and that aldosterone activates pcMTase without affecting transferase expression.

Phosphatidylinositol phosphate-dependent regulation of Xenopus ENaC by MARCKS protein

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.

Cathepsin B Is Secreted Apically from Xenopus 2F3 Cells and Cleaves the Epithelial Sodium Channel (ENaC) to Increase Its Activity

Background: Epithelial sodium channels (ENaC) are activated by proteolytic cleavage. Several proteases including furin and prostasin cleave ENaC. Results: Cathepsin B also cleaves and activates ENaC. Cathepsin B cleaves ENaC α but not β or γ subunits. Conclusion: Cathepsin B is a secreted protease, so it may cleave ENaC at the cell surface. Significance: Cathepsin B cleavage represents a novel ENaC regulatory mechanism. The epithelial sodium channel (ENaC) plays an important role in regulating sodium balance, extracellular volume, and blood pressure. Evidence suggests the α and γ subunits of ENaC are cleaved during assembly before they are inserted into the apical membranes of epithelial cells, and maximal activity of ENaC depends on cleavage of the extracellular loops of α and γ subunits. Here, we report that Xenopus 2F3 cells apically express the cysteine protease cathepsin B, as indicated by two-dimensional gel electrophoresis and mass spectrometry analysis. Recombinant GST ENaC α, β, and γ subunit fusion proteins were expressed in Escherichia coli and then purified and recovered from bacterial inclusion bodies. In vitro cleavage studies revealed the full-length ENaC α subunit fusion protein was cleaved by active cathepsin B but not the full-length β or γ subunit fusion proteins. Both single channel patch clamp studies and short circuit current experiments show ENaC activity decreases with the application of a cathepsin B inhibitor directly onto the apical side of 2F3 cells. We suggest a role for the proteolytic cleavage of ENaC by cathepsin B, and we suggest two possible mechanisms by which cathepsin B could regulate ENaC. Cathepsin B may cleave ENaC extracellularly after being secreted or intracellularly, while ENaC is present in the Golgi or in recycling endosomes.

WNK4 inhibition of ENaC is independent of Nedd4-2-mediated ENaC ubiquitination

A serine-threonine protein kinase, WNK4, reduces Na⁺ reabsorption and K⁺ secretion in the distal convoluted tubule by reducing trafficking of the thiazide-sensitive Na-Cl cotransporter to and enhancing renal outer medullary potassium channel retrieval from the apical membrane. Epithelial sodium channels (ENaC) in the distal nephron also play a role in regulating Na⁺ reabsorption and are also regulated by WNK4, but the mechanism is unclear. In A6 distal nephron cells, transepithelial current measurement and single channel recording show that WNK4 inhibits ENaC activity. Analysis of the number of channel per patch shows that WNK4 reduces channel number but has no effect on channel open probability. Western blots of apical and total ENaC provide additional evidence that WNK4 reduces apical as well as total ENaC expression. WNK4 enhances ENaC internalization independent of Nedd4-2-mediated ENaC ubiquitination. WNK4 also reduced the amount of ENaC available for recycling but has no effect on the rate of transepithelial current increase to forskolin. In contrast, Nedd4-2 not only reduced ENaC in the recycling pool but also decreased the rate of increase of current after forskolin. WNK4 associates with wild-type as well as Liddles mutated ENaC, and WNK4 reduces both wild-type and mutated ENaC expressed in HEK293 cells.

Cytosolic Phospholipase A2 Is Required for Optimal ATP Activation of BK Channels in GH3 Cells

To test the hypothesis that ATP activation of BK channels in GH3 cells involves cytosolic phospholipase A2 (cPLA2) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA2 by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA2, phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5′-adenylyl-β,γ-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA2-α (but not cPLA2-γ) as one target protein for phosphorylation that is intimately associated with the BK channel protein.

The sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC) associate

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.