He-Ping Ma

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.

Hydrogen Sulfide Prevents Hydrogen Peroxide-Induced Activation of Epithelial Sodium Channel through a PTEN/PI(3,4,5)P3 Dependent Pathway

Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC PO; however, in these cells H2O2 failed to elevate ENaC PO. Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.

Acute Regulation of Epithelial Sodium Channel by Anionic Phospholipids

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 plasma membrane, where these anionic phospholipids can regulate transmembrane proteins, including ion channels and transporters. Recent work has demonstrated that (1) ATP inhibits the renal epithelial sodium channel (ENaC) via a phospholipase C-dependent pathway that reduces PIP(2), (2) aldosterone stimulates ENaC via phosphoinositide 3-kinase, and (3) PIP(2) and PIP(3) regulate ENaC. Several lines of evidence show that ATP stimulation of purinergic P2Y receptors hydrolyzes PIP(2) and that aldosterone stimulation of steroid receptors induces PIP(3) formation. These studies together suggest that one primary mechanism for regulating ENaC is by alteration of anionic phospholipids and that the receptor-mediated and hormonal regulation of ENaC works through a variety of signaling pathways, but many of these pathways finally alter ENaC activity by regulating the formation or degradation of anionic phospholipids. Therefore, changes in the concentration of PIP(2) and PIP(3) are hypothesized to participate in the regulation of ENaC by purinergic and corticoid receptors. The underlying mechanism may be associated with a physical interaction of the positively charged cytoplasmic domains of the beta- and gamma-ENaC with the negatively charged membrane phospholipids. The exact nature of this interaction will require further investigation.

Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H2O2). In the cell-attached configuration, H2O2 significantly increased ENaC open probability (Po) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H2O2 are required to elevate intracellular H2O2, probably because catalase, the enzyme that promotes the decomposition of H2O2 to H2O and O2, is highly expressed in A6 cells. The effect of H2O2 on ENaC Po was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H2O2 levels are critical to produce the effect. However, H2O2 did not directly activate ENaC in inside-out patches. The effects of H2O2 on ENaC Po and amiloride-sensitive Na+ current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H2O2 elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P3, these data suggest that H2O2 stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P3.

High glucose induces podocyte apoptosis by stimulating TRPC6 via elevation of reactive oxygen species

Podocyte number is significantly reduced in diabetic patients and animal models, but the mechanism remains unclear. In the present study, we found that high glucose induced apoptosis in control podocytes which express transient receptor potential canonical 6 (TRPC6) channels, but not in TRPC6 knockdown podocytes in which TRPC6 was knocked down by TRPC6 silencing short hairpin RNA (shRNA). This effect was reproduced by treatment of podocytes with the reactive oxygen species (ROS), hydrogen peroxide (H2O2). Single-channel data from cell-attached, patch-clamp experiments showed that both high glucose and H2O2 activated the TRPC6 channel in control podocytes, but not in TRPC6 knockdown podocytes. Confocal microscopy showed that high glucose elevated ROS in podocytes and that H2O2 reduced the membrane potential of podocytes and elevated intracellular Ca(2+) via activation of TRPC6. Since intracellular Ca(2+) overload induces apoptosis, H2O2-induced apoptosis may result from TRPC6-mediated elevation of intracellular Ca(2+). These data together suggest that high glucose induces apoptosis in podocytes by stimulating TRPC6 via elevation of ROS.

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.

The A563T variation of the renal epithelial calcium channel TRPV5 among African Americans enhances calcium influx

The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) gene, which encodes the Ca(2+) channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits an unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer (45)Ca(2+) influx assay and the voltage-clamp technique using Xenopus laevis oocytes. Among the variations tested, including A8V, R154H, A563T, and L712F, the latter two significantly increased TRPV5-mediated Ca(2+) influx. The A563T variant, which exists in African Americans with relative high frequency, exhibited increased Ca(2+) influx at extracellular Ca(2+) from 0.01 to 2 mM despite a lower expression level at the plasma membrane. This variant also exhibited a reduction in Na(+) current as a result of increased sensitivity to extracellular Mg(2+). By substituting threonine-563 (Thr(563)) with serine or valine residue, the bulky side chain of Thr(563) was shown to facilitate Ca(2+) transport, whereas the hydroxyl group of Thr(563) is likely related to Mg(2+) sensitivity. The A563T variant was capable of increasing TRPV5-mediated Ca(2+) influx, even when it was expressed under conditions mimicking heterozygous or compound state with other variants. In conclusion, the A563T variant of TRPV5 significantly increased Ca(2+) influx by affecting the Ca(2+) permeation pathway. Thus the A563T variation in TRPV5 may contribute to the superior ability of renal Ca(2+) conservation in African Americans.

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.

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.