Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with ³⁵S or ³²P. When immune complexes were resolved on SDS-PAGE, the -subunit migrated at 85 and 65 kDa, the -subunit at 115 and 100 kDa, and the -subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The -subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of -, -, and -subunits approximately twofold. Exposure to 1 µM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport. epithelial sodium channel; aldosterone; sodium transport     

Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties

Endogenous serine proteases have been reported to control the reabsorption of Na(+) by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na(+) transport mediated by the epithelial Na(+) channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 +/- 10.5%) in the amiloride-sensitive sodium transport (I(Na)) with a time constant of 18 min and half maximal inhibition constant of 1 microM. Analysis of amiloride analogue blocker-induced fluctuations in I(Na) showed linear rate-concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947-C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of I(Na). A 50% decrease in I(Na) was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of I(Na) as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of I(Na) is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na(+) channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain I(Na) at some minimal threshold value.     

Syntaxin 1A regulates ENaC channel activity

Na(+) entry across the apical membranes of many absorptive epithelia is determined by the number (N) and open probability (P(o)) of epithelial sodium channels (ENaC). Previous results showed that the H3 domain of syntaxin-1A (S1A) binds to ENaC to reduce N, supporting a role for S1A in the regulation of ENaC trafficking. The aim of this study was to determine whether S1A-induced reductions in ENaC current also result from interactions between cell surface ENaC and S1A that alter ENaC P(o). Injection of a glutathione S-transferase (GST)-H3 S1A fusion protein into ENaC-expressing Xenopus oocytes inhibited whole cell Na(+) current (I(Na)) by 33% within 5 min. This effect was dose-dependent, with a K(i) of 7 ng/microl (approximately 200 nm). In contrast, injection of GST alone or a H3 domain-deleted GST-S1A fusion protein had no effect on I(Na). In cell-attached patch clamp experiments, GST-H3 acutely decreased ENaC P(o) by 30%, whereas GST-S1A Delta H3 was without effect. Further analysis revealed that ENaC mean closed time was significantly prolonged by S1A. Interestingly, GST-H3 had no effect on channel activity of an ENaC pore mutant that constitutively gates open (P(o) approximately equal 1.0), supporting the idea that S1A alters the closed state of ENaC and indicating that the actions of S1A on ENaC trafficking and gating can be separated experimentally. This study indicates that, in addition to a primary effect on ENaC trafficking, S1A interacts with cell surface ENaC to rapidly decrease channel gating. This rapid effect of S1A may modulate Na(+) entry rate during rapid increases in ENaC N.     

Regulation of TRPC6 channel activity by tyrosine phosphorylation

Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.     

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.     

Calmodulin-dependent regulation of hypotonicity-induced translocation of ENaC in renal epithelial A6 cells

Hypotonicity stimulates translocation of epithelial Na(+) channel (ENaC) to the apical membrane from the intracellular store site of ENaC by activating protein tyrosine kinase (PTK) in renal epithelial A6 cells. Based upon the fact that calmodulin shows its action on other enzymes through PTK caused phosphorylation of tyrosine residues of calmodulin itself, we studied whether a calmodulin-dependent pathway is involved in the action of hypotonicity on ENaC. W7, an antagonist of calmodulin, diminished the stimulatory action of hypotonicity on ENaC, irrespective of W7 treatment before or after application of hypotonicity. Calmodulin is known to regulate three pathways: (1) protein phosphatase 2B (PP2B), (2) Ca(2+)/calmodulin-dependent protein kinase II (CaMK II), and (3) myosin light chain kinase (MLCK). Pretreatment with cyclosporin A, an inhibitor of PP2B, did not influence the hypotonicity action on ENaC. The hypotonicity action on ENaC was partially inhibited by pretreatment with KN93, an inhibitor of CaMK II, but not by addition of KN93 after hypotonic stimulation had been applied. ML-7, an inhibitor of MLCK, showed the action similar to KN93. These observations indicate that: (1) the hypotonicity-induced translocation of ENaC depends on CaMK II and MLCK and (2) ENaC translocated to the apical membrane by hypotonicity is maintained in its activity and/or stability at the apical membrane through a calmodulin-dependent pathway.     

Cyclic GMP-activated channel activity in renal epithelial cells (A6).

We studied the effects of guanosine 3',5'-cyclic monophosphate (cGMP) and nitroprusside on ion channels in the apical membrane of confluent A6 cells (a distal nephron cell line) cultured on permeable supports for 10-14 days using patch clamp techniques. In cell-attached patches without any detectable channel activity, activity of a non-selective cation channel with a single-channel conductance of 1 pS was observed after adding nitroprusside. After adding cGMP to the cytosolic surface of inside-out patches with no detectable channel activity, we observed single channel activity similar to the channel observed after adding nitroprusside. These observations imply that nitroprusside activates a non-selective cation channel with small single channel conductance (1 pS) via an increase in cGMP which activates the channel.     

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.     

Regulation of sodium channel activity by phosphorylation

Voltage-gated sodium channels carry the major inward current responsible for action potential depolarization in excitable cells as well as providing additional inward current that modulates overall excitability. Both their expression and function is under tight control of protein phosphorylation by specific kinases and phosphatases and this control is particular to each type of sodium channel. This article examines the impact and mechanism of phosphorylation for isoforms where it has been studied in detail in an attempt to delineate common features as well as differences.     

Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells

Sodium absorption by an amiloride-sensitive channel is the main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. In this study, we tested whether tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine involved in several lung pathologies, could modulate sodium absorption in cultured alveolar epithelial cells. We found that TNF-alpha decreased the expression of the alpha-, beta-, and gamma-subunits of epithelial sodium channel (ENaC) mRNA to 36, 43, and 16% of the controls after 24-h treatment and reduced to 50% the amount of alpha-ENaC protein in these cells. There was no impact, however, on alpha(1) and beta(1) Na(+)-K(+)-ATPase mRNA expression. Amiloride-sensitive current and ouabain-sensitive Rb(+) uptake were reduced, respectively, to 28 and 39% of the controls. A strong correlation was found at different TNF-alpha concentrations between the decrease of amiloride-sensitive current and alpha-ENaC mRNA expression. All these data show that TNF-alpha, a proinflammatory cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport sodium.     

The effect of tetramethylammonium on single sodium channel currents.

Voltage-dependent Na conductance of rat myotubes was studied by patch recordings of single-channels. The patches were excised from the cell with the patch electrode, and the cytoplasmic surface was bathed in either CsF or tetramethylammonium (TMA)-F. Inward currents were examined from -20 to -50 mV. In this range Cs and TMA both appeared to be nearly impermeant, but TMA blocked the channel in a voltage-dependent manner. A first-order blocking site was located a maximum of 89% of the way through the membrane field from the cytoplasmic surface.     

Chronic exposure to EGF affects trafficking and function of ENaC channel in cystic fibrosis cells

Using the whole-cell patch-clamp technique, we identified an amiloride (AMI)-sensitive Na(+) current in cystic fibrosis cells, JME/CF15, growing in standard medium. The reversal potential of this current depended on Na(+) concentrations and the cation selectivity was much higher for Na(+) than for K(+), indicating that the current is through ENaC channels. In contrast, cells from EGF-containing medium lacked AMI-sensitive Na(+) currents. In permeabilized cells growing in EGF-containing medium, alphaENaC was mainly detected in a perinuclear region, while in cells from standard medium it was distributed over the cell body. Western-blot analysis showed that in standard medium cells expressed fast-migrating EndoH-insensitive and slow-migrating EndoH-sensitive alphaENaC fractions, while in cells growing in the presence of EGF, alphaENaC was only detected as the fast-migrating EndoH-insensitive fraction. Long-term incubation of cells with EGF resulted in an increased basal Ca(2+) level, [Ca(2+)](i). A similar increase of [Ca(2+)](i) was also observed in the presence of 2muM thapsigargin, resulting in inhibition of ENaC function. Thus, in JME/CF15 cells inhibition of the ENaC function by chronic incubation with EGF is a Ca(2+)-mediated process that affects trafficking and surface expression of ENaC channels.     

Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.     

Insulin-stimulated trafficking of ENaC in renal cells requires PI 3-kinase activity

ENaC-EGFP (enhanced green fluorescentprotein-tagged -subunit of the epithelial Na⁺ channel)stably transfected clonal lines derived from the A6 parental cell linewere used to study the physical mechanisms of insulin-stimulatedNa⁺ transport. Within 1 min of insulin stimulation, ENaCmigrates from a diffuse cytoplasmic localization to the apical andlateral membranes. Concurrently, after insulin stimulation,phosphatidylinositol 3-kinase (PI 3-kinase) is colocalized with ENaC onthe lateral but not apical membrane. An inhibitor of PI 3-kinase,LY-294002, does not inhibit ENaC/PI 3-kinase colocalization but doesalter the intracellular site of the colocalization, preventing thetranslocation of ENaC to the lateral and apical membranes. These datashow that insulin stimulation causes the migration of ENaC to thelateral and apical cell membranes and that this trafficking isdependent on PI 3-kinase activity.

     

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.     

The effect of retinoic acid on protein phosphorylation in mouse melanoma cells

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.     

The effect of antimycin A on mouse liver inner mitochondrial membrane channel activity.

In a patch-clamp study, we found antimycin A in low (1-2) microM concentrations decreased the open probability of the multiple conductance channel activity and the approximately 110 picosiemens channel of the inner mitochondrial membrane (for a review of mitochondrial channels see Kinnally, K. W., Antonenko, Yu. N., and Zorov, D. B. (1992) J. Bioenerg. Biomembr. 24, 99-110). Higher antimycin A concentrations (e.g. 10 microM) facilitated multiple conductance channel opening. These effects were reversible, and the binding site(s) are probably distinct from those responsible for the inhibition of the electron transport chain, since the latter are virtually irreversible. A model with two closed and two open states is presented for the approximately 110-picosiemens activity.     

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3beta (GSK-3beta) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.