Aldosterone induces ras methylation in A6 epithelia

Aldosterone increases Na⁺ reabsorption by renalepithelial cells: the acute actions (<4 h) appear to be promoted byprotein methylation. This paper describes the relationship betweenprotein methylation and aldosterone's action and describesaldosterone-mediated targets for methylation in cultured renal cells(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosteronestimulates protein methylation by increasing methyltransferase activityfrom 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce thealdosterone-induced increase in methyltransferase activity. One ofthese inhibitors, the isoprenyl-cysteine methyltransferase-specificinhibitor,S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone inducesprotein methylation in two molecular weight ranges: near 90 kDa andaround 20 kDa. The lower molecular weight range is the weight of smallG proteins, and aldosterone does increase both Ras protein 1.6-fold andRas methylation almost 12-fold. Also, Ras antisense oligonucleotidesreduce the activity of Na⁺ channels by about fivefold. Weconclude that 1) protein methylation is essential foraldosterone-induced increases in Na⁺ transport;2) one target for methylation is p21^ras; and3) inhibition of Ras expression or Ras methylation inhibits Na⁺ channel activity.

     

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

Weak channel blocker-induced noise analysis wasused to determine the way in which the steroids aldosterone andcorticosterone stimulated apical membraneNa⁺ entry into the cells oftissue-cultured A6 epithelia. Among groups of tissues grown on avariety of substrates, in a variety of growth media, and with cells atpassages 73-112, the steroidsstimulated both amiloride-sensitive and amiloride-insensitiveNa⁺ transport as measured byshort-circuit currents in chambers perfused with either growth mediumor a Ringer solution. From baseline rates of blocker-sensitiveshort-circuit current between 2 and 7 µA/cm², transport was stimulatedabout threefold in all groups of experiments. Single channel currentsaveraged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and weredecreased 6-20% from controls by steroid due to the expecteddecreases of fractional transcellular resistance. Irrespective ofbaseline transport rates, the steroids in all groups of tissuesstimulated transport by increase of the density of blocker-sensitiveepithelial Na⁺ channels (ENaCs).Channel open probability was the same in control and stimulatedtissues, averaging ~0.3 in all groups of tissues. Accordingly,steroid-mediated increases of open channel density responsible forstimulation of Na⁺ transport aredue to increases of the apical membrane pool of functional channels andnot their open probability.

     

sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+ transport

To studythe role of sgk (serum, glucocorticoid-induced kinase) inhormonal regulation of Na⁺ transport mediated by theepithelial Na⁺ channel (ENaC), clonal cell lines stablyexpressing human sgk, an S422A sgk mutant, or aD222A sgk mutant were created in the background of the A6model renal epithelial cell line. Expression of normal sgkresults in a 3.5-fold enhancement of basal transport and potentiationof the natriferic response to antidiuretic hormone (ADH). Transfectionof a S422A mutant form of sgk, which cannot bephosphorylated by phosphatidylinositol-dependent kinase (PDK)-2, results in a cell line that is indistinguishable from the parent linein basal and hormone-stimulated Na⁺ transport. The D222Asgk mutant, which lacks kinase activity, functions as adominant-negative mutant inhibiting basal as well as peptide- andsteroid hormone-stimulated Na⁺ transport. Thussgk activity is necessary for ENaC-mediated Na⁺transport. Phosphorylation and activation by PDK-2 are necessary forsgk stimulation of ENaC. Expression of normal sgkover endogenous levels results in a potentiated natriferic response toADH, suggesting that the enzyme is a rate-limiting step for the hormoneresponse. In contrast, sgk does not appear to be therate-limiting step for the cellular response to aldosterone or insulin.

     

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability     

Hormonal regulation of ENaCs: insulin and aldosterone

Although a variety of hormones and other agents modulate renalNa⁺ transport acting by way of theepithelial Na⁺ channel (ENaC), themode(s), pathways, and their interrelationships in regulation of thechannel remain largely unknown. It is likely that several hormones maybe present concurrently in vivo, and it is, therefore, important tounderstand potential interactions among the various regulatory factorsas they interact with the Na⁺transport pathway to effect modulation ofNa⁺ reabsorption in distal tubulesand other native tissues. This study represents specifically adetermination of the interaction between two hormones, namely,aldosterone and insulin, which stimulate Na⁺ transport by entirelydifferent mechanisms. We have used a noninvasive pulse protocol ofblocker-induced noise analysis to determine changes in single-channelcurrent (i_Na),channel open probability (P_o), andfunctional channel density(N_T) ofamiloride-sensitive ENaCs at various time points following treatmentwith insulin for 3 h of unstimulated control and aldosterone-pretreatedA6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs(N_T) involvedin transport. The very early (10 min) increases of channel density wereaccompanied by relatively small decreases ofi_Na(10-20%) and decreases ofP_o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes ofi_Na,P_o, andN_T weretransient, returning very slowly over 3 h toward their respectivecontrol values at the time of addition of insulin. We conclude thataldosterone and insulin act independently to stimulate apicalNa⁺ entry into the cells of A6epithelia by increase of channel density.

     

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

Several studies have shown that nitric oxide (NO) inhibits Na⁺ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na⁺ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug L-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 µM dexamethasone and 10% bovine serum. After 1.5 µM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound (P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability (P_o) from 0.186 ± 0.043 to 0.045 ± 0.009 (n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na⁺ transport is by altering iNOS production of NO. aldosterone; epithelial sodium channel; serum- and glycocorticoid-inducible kinase     

Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone

To examine the effect of aldosterone on sarcolemmalNa⁺ transport, we measuredouabain-sensitive electrogenicNa⁺-K⁺pump current(I_p) involtage-clamped ventricular myocytes and intracellularNa⁺ activity(aⁱ_Na) in right ventricularpapillary muscles. Aldosterone (10 nM) induced an increase in bothI_p and the rateof rise of aⁱ_Na duringNa⁺-K⁺pump blockade with the fast-acting cardiac steroid dihydroouabain. Thealdosterone-induced increase inI_p and rate ofrise of aⁱ_Na was eliminated bybumetanide, suggesting that aldosterone activates Na⁺ influx through theNa⁺-K⁺-2Clcotransporter. To obtain independent support for this, theNa⁺,K⁺, andCl concentrations in thesuperfusate and solution of pipettes used to voltage clamp myocyteswere set at levels designed to abolish the inward electrochemicaldriving force for theNa⁺-K⁺-2Clcotransporter. This eliminated the aldosterone-induced increase inI_p. We concludethat in vitro exposure of cardiac myocytes to aldosterone activates theNa⁺-K⁺-2Clcotransporter to enhance Na⁺influx and stimulate theNa⁺-K⁺pump.

     

A possible role for membrane depolarization in epithelial wound healing

Linear narrow wounds produced on cultured bovine corneal endothelial monolayers heal by actin cable formation at the wound border and lamellar crawling of cells into the injured area. We report the novel finding that membrane potential depolarization occurs at the leading edge of wounds and gradually extends inward toward the neighboring cells. We have determined that the replacement of extracellular Na⁺ by choline and the incorporation of phenamil, an inhibitor of the epithelial Na⁺ channel (ENaC), provoke a decrease in the actin cable and depolarization areas and in the lamellar activity of the wound edges. To the contrary, extracellular Li⁺ can successfully replace Na⁺ in the determination of the depolarization and cytoskeletal responses. This finding supports the idea that membrane depolarization, not the increase in intracellular Na⁺ concentration, is responsible for the formation of the actin cable, a result that is in agreement with previous evidence showing that nonspecific depolarization of the plasma membrane potential (PMP) of epithelial cells may promote characteristic cytoskeletal rearrangements per se (Chifflet S, Hernández JA, Grasso S, and Cirillo A. Exp Cell Res 282: 1–13, 2003). We suggest that spontaneous depolarization of the PMP of the cells at the wound borders determined by a rise in the ENaC activity of these cells constitutes an additional factor in the intermediate cellular processes leading to wound healing in some epithelia. actin; epithelial sodium channel     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

Inositol polyphosphate derivative inhibits Na+ transport and improves fluid dynamics in cystic fibrosis airway epithelia

Amiloride-sensitive, epithelial Na⁺ channel (ENaC)-mediated, active absorption of Na⁺ is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na⁺ and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and ²²Na⁺ flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives. epithelial Na⁺ channels; fluid absorption     

Glucocorticoids stimulate ENaC upregulation in bovine mammary epithelium

Mammary epithelia produce an isotonic, low-Na⁺ fluid that is rich in nutrients. Mechanisms that account for the low electrolyte concentration have not been elucidated, although amiloride-sensitive ion transport has been reported in some situations. We hypothesized that corticosteroid exposure modulates epithelial Na⁺ channel (ENaC) expression and/or activity in bovine mammary epithelial cells. BME-UV cells were grown to confluent monolayers on permeable supports with a standard basolateral medium and apical medium of low-electrolyte, high-lactose composition that resembles the ionic composition of milk. Ion transport was assessed in modified Ussing flux chambers. Exposure to glucocorticoids (dexamethasone, cortisol, or prednisolone), but not aldosterone, increased short-circuit current (I_sc), a sensitive measure of net ion transport, whereas apical exposure to amiloride or benzamil reduced corticosteroid-induced I_sc close to basal levels. Quantitative RT-PCR indicated a glucocorticoid-induced increase in mRNA for - and -ENaC, whereas -ENaC mRNA expression was only mildly affected. Exposure to mifepristone (a glucocorticoid receptor antagonist), but not spironolactone (a mineralocorticoid receptor antagonist), precluded both the corticosteroid-induced elevation in amiloride-sensitive I_sc and the induced changes in - and -ENaC mRNA. We conclude that Na⁺ movement across mammary epithelia is modulated by corticosteroids via a glucocorticoid receptor-mediated mechanism that regulates the expression of the - and -subunits of ENaC. ENaC expression and activity could account for the low Na⁺ concentration that is typical of milk. short-circuit current; apical cation concentration; corticosteroids; mastitis; epithelial Na⁺ channel subunits     

Role of Epithelial Sodium Channels and Their Regulators in Hypertension

The kidney has a central role in the regulation of blood pressure, in large part through its role in the regulated reabsorption of filtered Na⁺. Epithelial Na⁺ channels (ENaCs) are expressed in the most distal segments of the nephron and are a target of volume regulatory hormones. A variety of factors regulate ENaC activity, including several aldosterone-induced proteins that are present within an ENaC regulatory complex. Proteases also regulate ENaC by cleaving the channel and releasing intrinsic inhibitory tracts. Polymorphisms or mutations within channel subunits or regulatory pathways that enhance channel activity may contribute to an increase in blood pressure in individuals with essential hypertension.     

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells

Rapamycin and FK-506 are immunosuppressive drugs thatbind a ubiquitous immunophilin, FKBP12, but immunosuppressivemechanisms and side effects appear to be different. Rapamycin bindsrenal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na⁺ channels in A6cells. Channel NP_o was 0.5 ± 0.08 (n = 6)during the first 5 min but fell close to zero after 20 min. Application of 1 µM rapamycin reactivated Na⁺ channels(NP_o = 0.47 ± 0.1; n=6), but 1 µMFK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor,mimicked the rapamycin-induced reactivation in a nonadditive manner.However, rapamycin did not reactivate Na⁺ channels if cellswere exposed to 1 µM FK-506 before rapamycin. In PKC assays,rapamycin was as effective as the PKC inhibitor; however, epithelialNa⁺ channel (ENaC) phosphorylation was low under baselineconditions and was not altered by PKC inhibitors or activators. Theseresults suggest that rapamycin activates Na⁺ channels bybinding FKBP12 and inhibiting PKC, and, in renal cells, despite bindingthe same immunophilin, rapamycin and FK-506 activate differentintracellular signaling pathways.

     

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia

To study and define the early time-dependent response (6 h) ofblocker-sensitive epithelial Na⁺channels (ENaCs) to stimulation ofNa⁺ transport by aldosterone, weused a new modified method of blocker-induced noise analysis todetermine the changes of single-channel current (i_Na) channel open probability(P_o), andchannel density(N_T) undertransient conditions of transport as measured by macroscopic short-circuit currents(I_sc). In threegroups of experiments in which spontaneous baseline rates of transportaveraged 1.06, 5.40, and 15.14 µA/cm², stimulation of transportoccurred due to increase of blocker-sensitive channels.N_T variedlinearly over a 70-fold range of transport (0.5-35µA/cm²). Relatively small andslow time-dependent but aldosterone-independent decreases ofP_o occurredduring control (10-20% over 2 h) and aldosterone experimentalperiods (10-30% over 6 h). When theP_o of control andaldosterone-treated tissues was examined over the 70-fold extendedrange of Na⁺ transport,P_o was observedto vary inversely withI_sc, falling from~0.5 to ~0.15 at the highest rates ofNa⁺ transport or ~25% per3-fold increase of transport. Because decreases ofP_o from anysource cannot explain stimulation of transport by aldosterone, it isconcluded that the early time-dependent stimulation ofNa⁺ transport in A6 epithelia isdue exclusively to increase of apical membraneN_T.

     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex

Hormone regulation of ion transport in the kidney tubules is essential for fluid and electrolyte homeostasis in vertebrates. A large body of evidence has suggested that transporters and channels exist in multiprotein regulatory complexes; however, relatively little is known about the composition of these complexes or their assembly. The epithelial sodium channel (ENaC) in particular is tightly regulated by the salt-regulatory hormone aldosterone, which acts at least in part by increasing expression of the serine-threonine kinase SGK1. Here we show that aldosterone induces the formation of a 1.0–1.2-MDa plasma membrane complex, which includes ENaC, SGK1, and the ENaC inhibitor Nedd4-2, a key target of SGK1. We further show that this complex contains the PDZ domain-containing protein connector enhancer of kinase suppressor of Ras isoform 3 (CNK3). CNK3 physically interacts with ENaC, Nedd4-2, and SGK1; enhances the interactions among them; and stimulates ENaC function in a PDZ domain-dependent, aldosterone-induced manner. These results strongly suggest that CNK3 is a molecular scaffold, which coordinates the assembly of a multiprotein ENaC-regulatory complex and hence plays a central role in Na⁺ homeostasis.     

Regulation of epithelial Na+ channels (ENaC) by methylation: a novel methyltransferase stimulates ENaC activity

Aldosterone acts to increase apical membrane permeability by activation of epithelial Na(+) channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the beta subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that beta mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na(+) transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na(+) transport induced by aldosterone at early time points (1-4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. beta ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.     

The epithelial sodium channel and the control of sodium balance

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na⁺ absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure.Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na⁺ absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na⁺ absorption in the ASDN, and in the amount of Na⁺ that is excreted in the final urine.A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems.These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na⁺ in response to Na⁺ diet occurs predominantly in the early part (the connecting tubule) of the ASDN.An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na⁺ absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na⁺ balance.