Hui-Fang Bao

Differential Effects of Protein Kinase C on the Levels of Epithelial Na+ Channel Subunit Proteins

Regulation of epithelial Na+channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both β and γ, but not αENaC. Temporal regulation of β and γENaC by PKC differed; γENaC decreased with a time constant of 3.7 ± 1.0 h, whereas βENaC decreased in 13.9 ± 3.0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na+reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both β and γENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na+ transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of β and γ but not αENaC protein. These results imply a PKC-dependent, long term decrease in Na+ reabsorption.

Regulation of Epithelial Sodium Channel Trafficking by Ubiquitination

Amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC) play a crucial role in Na(+) transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na(+) transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends upon a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits, alpha, beta, and gamma. The C-terminal domain of all three subunits is intracellular and contains a proline rich motif (PPxY). Mutations or deletion of this PPxY motif in the beta and gamma subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein (Nedd4-2) to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when MDCK cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected epithelial cells (A6) expressing endogenous ENaC, ENaC appears to be degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by the rate of Nedd4-2-mediated ENaC ubiquitination. Controlling the rate of degradation is apparently important enough to have multiple, redundant pathways to control Nedd4-2 and ENaC ubiquitination.

ENaC activity is increased in isolated, split-open cortical collecting ducts from protein kinase Cα knockout mice

The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKCα influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKCα⁻/⁻ mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKCα, the open probability (P₀) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 ± 0.04 vs. 0.17 ± 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of α-, β-, and γ-subunits of ENaC in principal cells in the cortical collecting ducts of PKCα⁻/⁻ mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKCα⁻/⁻ mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and P₀ by PKCα in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.

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.

WNK1 Activates Large-Conductance Ca2+-Activated K+ Channels through Modulation of ERK1/2 Signaling

With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca(2+)-activated K(+) (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the α subunit of BK (HEK-BKα cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKα channel activity and open probability. Knockdown of WNK1 expression also significantly inhibited BKα protein expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKα expression and decreased ERK1/2 phosphorylation in a dose-dependent manner in HEK293 cells. Knockdown of ERK1/2 prevented WNK1 siRNA-mediated inhibition of BKα expression. Similarly, pretreatment of HEK-BKα cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of WNK1 siRNA on BKα expression in a dose-dependent manner. Knockdown of WNK1 expression also increased the ubiquitination of BKα channels. Notably, mice fed a high-K(+) diet for 10 days had significantly higher renal protein expression levels of BKα and WNK1 and lower levels of ERK1/2 phosphorylation compared with mice fed a normal-K(+) diet. These data suggest that WNK1 enhances BK channel function by reducing ERK1/2 signaling-mediated lysosomal degradation of the channel.

Ethanol stimulates epithelial sodium channels by elevating reactive oxygen species

Alcohol affects total body sodium balance, but the molecular mechanism of its effect remains unclear. We used single-channel methods to examine how ethanol affects epithelial sodium channels (ENaC) in A6 distal nephron cells. The data showed that ethanol significantly increased both ENaC open probability (P(o)) and the number of active ENaC in patches (N). 1-Propanol and 1-butanol also increased ENaC activity, but iso-alcohols did not. The effects of ethanol were mimicked by acetaldehyde, the first metabolic product of ethanol, but not by acetone, the metabolic product of 2-propanol. Besides increasing open probability and apparent density of active channels, confocal microscopy and surface biotinylation showed that ethanol significantly increased α-ENaC protein in the apical membrane. The effects of ethanol on ENaC P(o) and N were abolished by a superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) and blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Consistent with an effect of ethanol-induced reactive oxygen species (ROS) on ENaC, primary alcohols and acetaldehyde elevated intracellular ROS, but secondary alcohols did not. Taken together with our previous finding that ROS stimulate ENaC, the current results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde.

Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane.

Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells

We and others have shown that epithelial Na(+) channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patch-clamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.

Pendrin gene ablation alters ENaC subcellular distribution and open probability

The present study explored whether the intercalated cell Cl(-)/HCO3(-) exchanger pendrin modulates epithelial Na(+) channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na(+) absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddles syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddles syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddles syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability.