Knockout of mitochondrial voltage-dependent anion channel type 3 increases reactive oxygen species (ROS) levels and alters renal sodium transport.

It has been suggested that voltage-dependent anion channels (VDACs) control the release of superoxide from mitochondria. We have previously shown that reactive oxygen species (ROS) such as superoxide (O2̇̄) and hydrogen peroxide (H2O2) stimulate epithelial sodium channels (ENaCs) in sodium-transporting epithelial tissue, including cortical collecting duct (CCD) principal cells. Therefore, we hypothesized that VDACs could regulate ENaC by modulating cytosolic ROS levels. Herein, we find that VDAC3-knockout(KO) mice can maintain normal salt and water balance on low-salt and high-salt diets. However, on a high-salt diet for 2 weeks, VDAC3-KO mice had significantly higher systolic blood pressure than wildtype mice. Consistent with this observation, after a high-salt diet for 2 weeks, ENaC activity in VDAC3-KO mice was significantly higher than wildtype mice. EM analysis disclosed a significant morphological change of mitochondria in the CCD cells of VDAC3-KO mice compared with wildtype mice, which may have been caused by mitochondrial superoxide overload. Of note, compared with wildtype animals, ROS levels in VDAC3-KO animals fed a normal or high-salt diet were consistently and significantly increased in renal tubules. Both the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL) and the mitochondrial ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mito-TEMPO) could reverse the effect of high-salt on ENaC activity and systolic blood pressure in the VDAC3-KO mice. Mito-TEMPO partially correct the morphological changes in VDAC3-KO mice. Our results suggest that knocking out mitochondrial VDAC3 increases ROS, alters renal sodium transport, and leads to hypertension.

Mal protein stabilizes luminal membrane PLC-ß3 and negatively regulates ENaC in mouse cortical collecting duct cells.

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl- cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-ß3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-ß3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-ß3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

High glucose reduces expression of podocin in cultured human podocytes by stimulating TRPC6.

The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca2+ in control podocytes but not in TRPC6 knockdown podocytes. High glucose also elevated the expression of a tight junction protein, zonula occludens-1, and induced the redistribution of zonula occludens-1 and loss of podocyte processes. These data together suggest that high glucose reduces protein levels of podocin by activating TRPC6 and induces morphological changes of cultured podocytes.

G protein pathway suppressor 2 enhanced the renal large-conductance Ca2+-activated potassium channel expression via inhibiting ERK1/2 signaling pathway.

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+-activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity ( NPo), increasing BK open probability ( Po), and channel number ( N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli.

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. METHODS: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. RESULTS: Here we show that extraction of membrane Cho with methyl-ß-cyclodextrin (MßCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP2 in the microvilli and that Cho was also co-localized with PIP2 in the microvilli. Either extraction of Cho with MßCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP2, and ENaC in the microvilli. CONCLUSIONS: Since PIP2 can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP2 in the microvilli.

ENaC activity is regulated by calpain-2 proteolysis of MARCKS proteins.

We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca2+-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca2+ Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca2+ mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.

The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria.

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.

Unoprostone activation of BK (KCa1.1) channel splice variants.

This investigation was conducted to study the relationship between intracellular Ca(2+) and activation of large conductance Ca(2+)-activated K(+) (BK) currents by unoprostone, the first synthetic docosanoid. We used HEK293 cells stably transfected with two BK channel splice variants, one sensitive to unoprostone and the other insensitive. We examined the effects of unoprostone on channel activity in excised inside-out patches and cell-attached patches. The half-maximal stimulation of the sensitive BK channels by Ca(2+) was shifted from 3.4±0.017 nM to 0.81±.0058 nM in the presence of 10 nM unoprostone. There was no effect on insensitive channels even at unoprostone concentrations as high as 1000 nM. There was no effect of unoprostone on the voltage dependence of the BK channels. Changes in open probability and effects of Ca(2+) and unoprostone were best described by a synergistic binding model. These data would suggest that Ca(2+) and unoprostone were binding to sites close to one another on the channel protein and that unoprostone binding causes the affinity of the calcium binding site to increase. This idea is consistent with three dimensional models of the Ca(2+) binding site and a putative unoprostone binding domain. Our results have important implications for the clinical use of unoprostone to activate BK channels. Channel activation will be limited if intracellular Ca(2+) is not elevated.

Acute ethanol induces apoptosis by stimulating TRPC6 via elevation of superoxide in oxygenated podocytes.

Our recent studies indicate that hydrogen peroxide (H2O2) only at high concentrations can cause oxidative stress in renal epithelial cells and induce apoptosis of podocytes. Consistently, the present study shows that H2O2, even at 1 mM, failed to induce intracellular oxidative stress and apoptosis of the podocytes due to efficient activity of catalase, an enzyme which degrades H2O2 to produce water and oxygen (O2). However, H2O2 acted as a source of O2 to allow acute ethanol to induce superoxide production and cause apoptosis of the podocytes. In contrast, acute ethanol alone did not elevate intracellular superoxide, even though it stimulates expression and translocation of p47phox to the plasma membrane. Inhibition of catalase abolished not only O2 production from H2O2 degradation, but also NOX2-dependent superoxide production in the podocytes challenged by both H2O2 and acute ethanol. In parallel, acute ethanol in the presence of H2O2, but neither ethanol nor H2O2 alone, stimulated transient receptor potential canonical 6 (TRPC6) channels and caused TRPC6-dependent elevation of intracellular Ca2+. These data suggest that exogenous H2O2 does not induce oxidative stress due to rapid degradation to produce O2 in the podocytes, but the oxygenated podocytes become sensitive to acute ethanol challenge and undergo apoptosis via a TRPC6-dependent elevation of intracellular Ca2+. Since cultured podocytes are considered in hypoxic conditions, H2O2 may be used as a source of O2 to establish an ischemia-reperfusion model in some type of cultured cells in which H2O2 does not directly induce intracellular oxidative stress.

Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells.

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

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.

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

We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K(+) [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia.

Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression.

Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca(2+)-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca(2+) also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca(2+).

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.

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.

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-, ß-, 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.

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex.

Cathepsin B is secreted apically from Xenopus 2F3 cells and cleaves the epithelial sodium channel (ENaC) to increase its activity.

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, ß, 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 ß 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.

Rituximab inhibits Kv1.3 channels in human B lymphoma cells via activation of FcγRIIB receptors.

Kv1.3 channels play an important role in modulating lymphocyte proliferation and apoptosis. We hypothesized that Kv1.3 channels in B lymphocytes might be regulated by rituximab, an antibody to CD20, a drug for treatments of B-cell lymphomas and autoimmune diseases. Using both whole-cell and cell-attached patch-clamp techniques, we found that rituximab inhibited Kv1.3 channels in Daudi human B lymphoma cells by promoting the channel inactivation at a concentration which was much greater than that required for activation of CD20. The effect of rituximab on Kv1.3 channels was abolished after selective blockade of FcγRIIB receptors with anti-FcγRIIB antibody. Western blot experiments showed that Daudi B cells expressed both Kv1.3 channel and the low affinity Fc receptor, FcγRIIB, which could be activated by the Fc region of rituximab. In contrast, normal lymphocytes expressed less Kv1.3 channels with faster inactivation. Confocal microscopy and flow cytometry data showed that rituximab induced apoptosis of Daudi B cells and that the effect was attenuated by blockade of FcγRIIB receptors and partially mimicked by inhibition of Kv1.3 channels. These results suggest that in addition to previously described complement-dependent cytotoxicity, rituximab also induces apoptosis of malignant B lymphocyte by stimulating FcγRIIB receptors and inhibiting Kv1.3 channels.

Fulvene-5 inhibition of Nadph oxidases attenuates activation of epithelial sodium channels in A6 distal nephron cells.

Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel antioxidant drugs, such as Nox4 inhibitor compounds, are being developed. There is, however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single-channel patch-clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04 ± 0.18 (means ± SE) to 0.25 ± 0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic cell stretch response of A6 cells, indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertension.