Anionic phospholipids differentially regulate the epithelial sodium channel (ENaC) by interacting with alpha, beta, and gamma ENaC subunits.

Anionic phospholipids (APs) present a variety of lipids in the cytoplasmic leaflet of the plasma membrane, including phosphatidylinositol (PI), PI-4-phosphate (PI(4)P), phosphatidylserine (PS), PI-4,5-bisphosphate (PI(4,5)P(2)), PI-3,4,5-trisphosphate (PI(3,4,5)P(3)), and phosphatidic acid (PA). We previously showed that PI(4,5)P(2) and PI(3,4,5)P(3) upregulate the renal epithelial sodium channel (ENaC). Further studies from others suggested that PI(4,5)P(2) and PI(3,4,5)P(3) respectively target beta- and gamma-ENaC subunit. To determine whether PI(4,5)P(2) and PI(3,4,5)P(3) selectively bind to beta and gamma subunit, we performed lipid-protein overlay experiments. Surprisingly, the results reveal that most APs, including PI(4)P, PS, PI(4,5)P(2), PI(3,4,5)P(3), and PA, but not PI, non-selectively bind to not only beta and gamma but also alpha subunit. To determine how these APs regulate ENaC, we performed inside-out patch-clamp experiments and found that PS, but not PI or PI(4)P, maintained ENaC activity, that PI(4,5)P(2) and PI(3,4,5)P(3) stimulated ENaC, and that PA, however, inhibited ENaC. These data together suggest that APs differentially regulate ENaC by physically interacting with alpha-, beta-, and gamma-ENaC. Further, the data from cell-attached patch-clamp and confocal microscopy experiments indicate that PA, a product of phospholipase D, may provide one of the pathways for inhibition of ENaC by endothelin receptors.

Cyclosporine stimulates the renal epithelial sodium channel by elevating cholesterol.

Cyclosporine A (CsA) is an efficient immunosuppressant used for reducing allograft rejection but with a severe side effect of causing hypertension. We hypothesize that the renal epithelial sodium channel (ENaC) may participate in CsA-induced hypertension. In the present study, we used the patch-clamp cell-attached configuration to examine whether and how CsA stimulates ENaC in A6 distal nephron cells. The data showed that CsA significantly increased ENaC open probability. Since CsA is an inhibitor of the ATP-binding cassette A1 (ABCA1) transporter, we employed 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), another ABCA1 inhibitor, and found that DIDS mimicked the effects of CsA on ENaC basal and cholesterol-induced activity but without any additive effect if combined with CsA. CsA and DIDS also had an identical effect on reduced ENaC activity caused by cholesterol extraction. ABCA1 protein was detected in A6 cells by Western blot analysis. Confocal microscopy data showed that both CsA and DIDS facilitated A6 cells to uptake cholesterol. Since enhanced ENaC activity is known to cause hypertension, these data together suggest that CsA may cause hypertension by stimulating ENaC through a pathway associated with inhibition of ABCA1 and consequent elevation of cholesterol in the cells.

An intermediate-conductance Ca(2+)-activated K (+) channel mediates B lymphoma cell cycle progression induced by serum.

We have previously reported that Kv1.3 channel is expressed in Daudi cells. However, the present study demonstrates that Daudi cell cycle progression is not affected by margatoxin, a Kv1.3 channel blocker, but can be suppressed by tetraethylammonium (TEA) and 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), a selective blocker of intermediate-conductance Ca(2+)-activated K(+) (IK) channels. Our patch-clamp data indicate that Daudi cells express an IK channel because it has a unit conductance of about 30 pS, is voltage-independent, and can be activated by submicromolar Ca(2+) and blocked by TRAM-34. Fetal bovine serum (FBS) elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) and activated this IK channel. Conversely, Rituximab, a human-mouse chimeric monoclonal antibody of CD20, significantly decreased [Ca(2+)](i) and inhibited the channel. Furthermore, both FBS-induced IK channel expression and cell cycle progression were attenuated by the treatment with LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. These data together suggest that a growth factor(s) in FBS triggers cell cycle progression by elevating both IK channel activity via CD20 and IK channel expression on the cell surface via PI3K. Thus, elevated IK channel activity and expression may account, in part, for Daudi cell malignant growth and proliferation.

Ceramide mediates inhibition of the renal epithelial sodium channel by tumor necrosis factor-alpha through protein kinase C.

To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-alpha (TNF-alpha), confocal microscopy and patch-clamp experiments were performed in A6 distal nephron cells. We found that TNF-alpha (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-alpha was mimicked by exogenous sphingomyelinase (0.1 U/ml) and C(2)-ceramide (50 microM), but neither C(2)-dihydroceramide, a membrane-impermeable analog of C(2)-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. C(2)-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. C(2)-ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GF109203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-alpha inhibition of the renal ENaC via a pathway associated with PKC-dependent externalization of PS.

Membrane tension modulates the effects of apical cholesterol on the renal epithelial sodium channel.

We used patch-clamp techniques and A6 distal nephron cells as a model to determine how cholesterol regulates the renal epithelial sodium channel (ENaC). We found that luminal methyl-beta-cyclodextrin (mbetaCD, a cholesterol scavenger) did not acutely affect ENaC activity at a previously used concentration of 10 mM: but significantly decreased ENaC activity both when the cell membrane was stretched and at a higher concentration of 50 mM: Luminal cholesterol had no effect on ENaC activity at a concentration of 50 microg/ml but significantly increased ENaC activity both when the cell membrane was stretched and at a higher concentration of 200 microg/ml. Confocal microscopy data indicate that membrane tension facilitates both mbetaCD extraction of cholesterol and A6 cell uptake of exogenous cholesterol. Together with previous findings that cholesterol in the apical membrane is tightly packed with sphingolipids and that stretch can affect lipid distribution, our data suggest that membrane tension modulates the effects of mbetaCD and cholesterol on ENaC activity, probably by facilitating both extraction and enrichment of apical cholesterol.

Phosphatidylinositol 3,4,5-trisphosphate mediates aldosterone stimulation of epithelial sodium channel (ENaC) and interacts with gamma-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 (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 gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(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 plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway.

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl- for gluconate(-) shifted the reversal potential, while Cl- channel blockers, 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPgammaS (a poorly hydrolyzable analog of ATP), 2',3'-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 microM suramin (a P2 receptor antagonist), and was partially blocked by 100 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca(2+)-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.

Steroids and exogenous gamma-ENaC subunit modulate cation channels formed by alpha-ENaC in human B lymphocytes.

Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both alpha- and beta- but not gamma-ENaC. Western blots showed that both alpha- and beta- but not gamma-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against alpha-ENaC but not by antisense oligonucleotide against beta-ENaC, indicating that only the alpha subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 microm) reduced the frequency of observance of 21-pS alpha-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from alpha- and beta-ENaC. After expression of exogenous gamma-ENaC, aldosterone again reduced the frequency of observance of the 21-pS alpha-ENaC channel but induced the appearance of a 5-pS channel, presumably a alphabetagamma-ENaC channel. In the absence of aldosterone, the alpha subunit forms an alpha-cryptic channel that is activated by stretch, and in the presence of aldosterone, beta and alpha subunits together form an active channel that is modulated by aldosterone.