Rho Small GTPases Activate the Epithelial Na+ Channel*

Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na+ channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P2 levels promoting channel insertion into the plasma membrane.

The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is localized to the luminal plasma membrane of epithelial cells where its activity is rate-limiting for Na ϩ transport (1,2). Thus ENaC plays a pivotal role in Na ϩ and concomitant water (re)absorption across many epithelial tissues making it a centrally positioned effector for hormones and other factors that modulate systemic blood pressure and epithelial surface hydration (3,4). Although it is accepted that ENaC activity is dynamically controlled by regulation of channel localization to the luminal plasma membrane and channel gating, little is known about the cellular control points impinging upon this modulation.
Membrane phospholipids such as PI(4,5)P 2 influence the activity of ion channels, including ENaC, by influencing channel open probability and membrane levels (5)(6)(7). We have demonstrated previously that small G proteins within the Ras family (e.g. K-RasA) augment ENaC activity by stimulating PI3K signaling (8) with the phospholipid products of this lipid kinase (PI(3,4)P 2 and PI (3,4,5)P 3 ) increasing channel open probability. Regulation of ENaC by other small G proteins has not been reported.
RhoA and Rac1 are small, monomeric G proteins within the Rho family of GTPases (9,10). Similar to other small G proteins, these GTPases act as molecular switches cycling between active GTP-bound and inactive GDP-bound states. In the GTPbound state, Rho GTPases interact with effector molecules such as Rho kinase to initiate downstream responses. Both RhoA and Rac1 as well as Rho kinase increase PI(4,5)P 2 levels by activating PI(4)P 5-K (11)(12)(13). Through this phospholipid second messenger and other adapter proteins, Rho GTPases influence vesicle movement impacting endocytosis as well as exocytosis of integral plasma membrane proteins (14 -16).
Recently, Rho GTPase signaling has been shown to modulate activity of several classes of ion channels, including Kv1.2 and ERG K ϩ channels, Ca 2ϩ channels, and nonselective cation channels (17)(18)(19)(20). Moreover, Rho GTPase signaling increases the membrane levels of the transient receptor potential channel TRPC5 through a PI(4)P 5-K-dependent process (21). Thus, although the possibility that RhoA regulates ENaC activity has not been directly studied, a variety of ion channels are now beginning to be recognized as final effectors for RhoA and other GTPases within this family.
The current study asked whether Rho GTPases influence ENaC activity and whether phospholipid signaling was a component of this regulation. We find that RhoA and Rac1 increase ENaC activity with Rho kinase necessary for this activation. Similarly, the downstream RhoA-Rho kinase effector PI(4)P 5-K augments ENaC activity, and RhoA is unable to enhance ENaC activity when generation of PI(4,5)P 2 is disrupted. The major effect of RhoA on ENaC was to increase the plasma membrane levels of this channel. We conclude that RhoA-Rho kinase signaling stimulates PI(4)P 5-K to increase PI(4,5)P 2 levels with dependent increases in the plasma membrane levels of ENaC.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were of reagent grade and were purchased from either Calbiochem, BioMol (Plymouth Meeting, PA), or Sigma unless noted otherwise. The mammalian expression vectors encoding Myc-tagged and human ENaC subunit cDNAs have been described previously (7,22,23). Expression vectors encoding hemagglutinintagged wild type, constitutively active (G14V) and dominant negative (T19N) RhoA, and wild type Rac1 were from the Guthrie Research Institute. The mammalian expression vector encoding PI(4)P 5-K was a kind gift from Dr. M. S. Shapiro. All materials used in Western blot analysis were from Bio-Rad. Sulfo-NHS-LC-biotin and streptavidinagarose were from Pierce. The mouse monoclonal anti-Myc antibody was from Clontech (Palo Alto, CA). Anti-mouse horseradish peroxidaseconjugated secondary antibody was from Kirkegaard & Perry Laboratories (Gaithersburg, MD). ECL reagents were from PerkinElmer Life Sciences. CHO and COS-7 cells were maintained with standard culture conditions (Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, 37°C, 5% CO 2 ) and transfected using the Polyfect reagent (Qiagen, Valencia, CA) as described previously (7,22,23).
Membrane Labeling Experiments-Membrane labeling experiments closely followed those described previously (27). In brief, COS-7 cells were transfected with Myc-tagged ENaC in the absence and presence of wild type, constitutively active, and dominant negative RhoA. 48 h after transfection cells were washed three times with ice-cold phosphatebuffered saline (pH 8.0) and subsequently incubated with 1 mM sulfo-NHS-LC-biotin (in phosphate-buffered saline, pH 8.0) for 30 min at 4°C in the dark. Cells were washed three times with ice-cold phosphatebuffered saline and extracted in gentle lysis buffer (1.0% Nonidet P-40), cleared, and normalized for total protein concentration. Pre-equilibrated streptavidin-agarose beads were agitated overnight at 4°C with 100 g of total protein. Agarose beads were then washed six times with gentle lysis buffer and subsequently resuspended in SDS sample buffer and 20 mM dithiothreitol, heated at 85°C for 10 min, run on 7.5% polyacrylamide gels in the presence of SDS, transferred to nitrocellulose, and probed with anti-Myc antibody in Tris-buffered saline supplemented with 5% dried milk and 0.1% Tween 20.
Statistics-All patch clamp data are presented as mean Ϯ S.E. Paired and unpaired data were compared using appropriate t tests with p Յ 0.01 considered significant.

Rho GTPases Increase ENaC
Activity-To investigate the actions of Rho GTPases on ENaC, we focused on the two representative family members RhoA and Rac1. For these studies, we reconstituted human ENaC (hENaC) in CHO cells in the absence and presence of co-expressed wild type, constitutively active (G14V), and dominant negative (T19N) RhoA and wild type Rac1. hENaC was reconstituted by co-expressing ␣, ␤, and ␥ channel subunits together (0.5 g/35 mm 2 each cDNA). Fig.  1A shows a continuous recording of macroscopic Na ϩ currents evoked by a train of voltage ramps in a typical voltage clamp experiment performed on a CHO cell expressing ENaC before and after the addition of amiloride (10 M) and the subsequent washout of this channel inhibitor. Fig. 1B shows typical currents from voltage clamp experiments performed on CHO cells containing ENaC alone and with co-expression of the various RhoA GTPases. Currents were elicited by applying test pulses (20-mV increments) from Ϫ120 to 100 mV in the absence (Fig.  1B, left) and presence (right) of 10 M amiloride in the bathing solution. Overexpression of ENaC resulted in substantial amiloride-sensitive (IC 50 at Ϫ80 mV ϳ 200 nM), inward Na ϩ currents that were not present in untransfected cells (not shown, see Refs. 7 and 22). Co-expression of wild type and constitutively active but not dominant negative RhoA markedly increased ENaC activity. The average amiloride-sensitive current density at Ϫ80 mV, as shown in Fig. 1C, in control experiments (hENaC alone) was 173.9 Ϯ 17.4 pA/pF (n ϭ 14). Co-overexpression of wild type RhoA and constitutively active RhoA G14V with hENaC significantly increased current density to 437.2 Ϯ 82.0 pA/pF (n ϭ 11) and 671.0 Ϯ 65.2 pA/pF (n ϭ 8), respectively. These results are consistent with RhoA in the GTP-bound state enhancing ENaC activity. In contrast, coexpression of dominant negative RhoA T19N , which prevents activation of endogenous RhoA and its effectors by forming FIG. 1. Rho GTPases increase ENaC activity. A, typical macroscopic Na ϩ currents in a voltage clamp experiment evoked by a continuous train of voltage ramps applied every 5 s to a CHO cell expressing ENaC before and after treatment with amiloride and subsequent washout of channel inhibitor. Inward current is down. B, typical macroscopic current traces before (left) and after (right) amiloride (amil) under voltage clamp conditions from CHO cells transfected with human ENaC alone and with wild type, constitutively active (G14V), and dominant negative (T19N) RhoA. Currents elicited by test pulses with 20-mV steps from 100 to Ϫ120 mV from a holding potential of 30 mV. C, summary graph of the mean Ϯ S.E. amiloride-sensitive current density at Ϫ80 mV for voltage-clamped CHO cells expressing hENaC in the absence and presence of wild type, constitutively active (G14V), dominant negative (T19N) RhoA, and Rac1. The number of observations in each group is shown. *, p Ͻ 0.01 versus hENaC alone.
unproductive complexes with Rho GTP exchange factors and effector proteins (24), resulted in a statistically significant decrease in hENaC activity compared with control. Current density in this latter case was reduced 5.4 times to 32.5 Ϯ 5.8 pA/pF (n ϭ 12) indicating that dominant negative RhoA inhibits the actions of endogenous RhoA on hENaC.
We next tested whether the positive effects of RhoA on ENaC were specific or a general feature of Rho GTPases by co-expressing ENaC plus Rac1. Co-expression of Rac1 with ENaC similar to RhoA resulted in increased ENaC activity (549.9 Ϯ 85.7 pA/pF, n ϭ 8). Overexpression of RhoA and its dominant negative and constitutively active forms as well as Rac1 had no effect on cell capacitance (not shown). These results showing RhoA and Rac1 augmentation of ENaC activity suggest that this may be a common feature shared by several members of the Rho family.
We confirmed the effects of RhoA on ENaC using the C3 exoenzyme, which selectively ADP-ribosylates and thus inactivates Rho proteins. Ras, as well as other small G proteins outside the Rho family, are not targets for the C3 exoenzyme (25). Because the C3 exoenzyme is a protein that does not penetrate the cell plasma membrane, we added this protein directly to the intracellular patch pipette solution. Fig. 2 shows the inhibitory effect of 5 g/ml C3 exoenzyme in cells co-expressing hENaC and wild type RhoA. Current density de-creased from 454.0 Ϯ 101.7 to 101.8 Ϯ 25.8 pA/pF. In contrast, the C3 exoenzyme had little effect on ENaC activity in the absence of co-expression of RhoA.
RhoA Increases ENaC Activity in a GTP-sensitive Manner-We next tested whether RhoA actions on ENaC were dependent on GTP. For these experiments, 2.0 mM GDP␤S (a non-hydrolysable analogue of GDP) replaced the GTP in typical intracellular pipette solutions. Fig. 3A shows the effect of GDP␤S in a representative cell overexpressing ENaC and RhoA. As GDP␤S dialyzed the cytosol, ENaC activity decreased indicating that GTP is necessary for RhoA to enhance channel activity. Fig. 3B shows a summary graph of experiments testing the role of GTP in RhoA-dependent activation of ENaC.
RhoA Activation of ENaC Is Mediated by Rho Kinase Signaling-We next asked whether RhoA activated ENaC through signaling via one of its downstream effector proteins/cascades: Rho kinase and the MAPK and PI3K cascades. Our rationale was that RhoA most likely communicates with ENaC through a signaling cascade and Ras GTPases activate ENaC via the PI3K cascade, which is also known to be activated by Rho GTPases and to activate Rho GTPases. Fig. 4A shows overlays of hENaC currents from typical whole cell voltage clamp experiments elicited by standard voltage ramps before and after amiloride. Currents are from representative cells expressing hENaC plus RhoA in the absence (Fig. 4A, top) and presence (bottom) of pretreatment with Y27632 (1 M, 1 h), which is an inhibitor of Rho kinase (26,27). As summarized in Fig. 4B, pretreating cells overexpressing hENaC and RhoA with Y27632 significantly decreased RhoA-dependent ENaC activity from 484.6 Ϯ 66.9 to 128.0 Ϯ 33.7 pA/pF. In contrast, neither the MAPK inhibitor PD98059 (10 M, 5 h) nor the PI3K inhibitors wortmannin (200 nM, 5 h) and LY294002 (50 M, 5 h) influenced RhoA actions on ENaC. We conclude from these results that Rho kinase (but not PI3K and MAPK) is necessary for RhoA-dependent activation of ENaC suggesting that this RhoA effector is positioned between the small G protein and the channel.
PI(4,5)P 2 Is Necessary for RhoA-dependent Activation of ENaC-RhoA, which physically interacts with PI(4)P 5-K, stimulates the activity of this phospholipid kinase via Rho kinase (14,16,28). Thus we wondered whether activation of PI(4)P 5-K in response to RhoA-Rho kinase signaling played a role in enhancing ENaC activity. To test this possibility, we first asked whether overexpression of PI(4)P 5-K influenced ENaC in a manner similar to RhoA. Fig. 5A shows typical current traces before and after amiloride from representative cells expressing hENaC alone and with PI(4)P 5-K. All conditions are the same as Fig. 1 with currents elicited by voltage

FIG. 2. C3 exoenzyme attenuates activation of ENaC by RhoA.
Summary graph of the amiloride-sensitive current density at Ϫ80 mV for hENaC alone and with co-expression of RhoA in the absence and presence of C3 exoenzyme (5 g/ml) included in the intracellular pipette solution. All conditions are the same as in Fig. 1. *, p Ͻ 0.01 versus ENaC alone; **, p Ͻ 0.01 versus ENaC plus RhoA.

FIG. 3. RhoA activates ENaC in a GTP-dependent manner.
A, overlay of typical macroscopic current traces from a representative CHO cell co-expressing hENaC plus RhoA at the beginning of the experiment (start) and 1 and 2 min after dialyzing the cytosol with 2 mM GDP␤S. Amiloride (amil) was added at the end of the experiment. Currents elicited by voltage ramps stepping from a holding potential of 30 to 60 mV and ramping to Ϫ100 mV. B, summary graph of the amiloridesensitive current density for hENaC plus RhoA in the absence and presence of GDP␤S in pipette solution. *, p Ͻ 0.01 versus hENaC plus RhoA. ramps. As summarized in Fig. 5B, co-expression of PI(4)P 5-K with ENaC significantly enhanced channel activity from 158.0 Ϯ 32.9 to 588.7 Ϯ 83.5 pA/pF. These results demonstrate that similar to RhoA, overexpression of its downstream effector PI(4)P 5-K augments ENaC activity suggesting that this effector is positioned between the small G protein and the channel and that the phospholipid products of this kinase transduce the stimulatory signal to ENaC.
To further probe whether PI(4)P 5-K and its phospholipid product PI(4,5)P 2 play a role in RhoA-dependent activation of ENaC, we tested the effects of this small G protein on the channel in the presence of inhibited PI4K, which precludes PI(4,5)P 2 synthesis. Fig. 6 summarizes ENaC activity in the absence and presence of co-expressed RhoA in cells not pretreated and those pretreated with 50 M wortmannin for 30 min to inhibit PI4K. At this dose and treatment time, wortmannin inhibits both PI3K and PI4K; however, our results shown in Fig. 4 excluded PI3K from playing a role in RhoAmediated effects on ENaC, and thus wortmannin was used to test the role of PI4K and dependent synthesis of PI(4,5)P 2 on RhoA-dependent activation of ENaC. Channel activity when ENaC was expressed alone decreased 3.2-fold from 123.3 Ϯ 22.7 to 38.5 Ϯ 7.3 pA/pF with wortmannin pretreatment. When expressed with RhoA, channel activity decreased 5.5-fold from 482.0 Ϯ 44.8 to 88.1 Ϯ 34.0 pA/pF with wortmannin pretreatment. These results clearly show that PI(4,5)P 2 synthesis is necessary for RhoA-dependent activation of the channel with this phospholipid either being permissive for RhoA effects or downstream of the small GTPase in its signal transduction cascade to the channel. We believe the latter is more likely considering the results shown in Fig. 5  others documenting RhoA-and Rho kinase-dependent activation of PI(4)P 5-K and associated increases in PI(4,5)P 2 synthesis (11,28).
RhoA Increases ENaC Activity by Increasing the Number of Channels in the Plasma Membrane-As mentioned above, PI(4,5)P 2 is known to influence vesicle movement and facilitate insertion/retention of integral membrane proteins in the plasma membrane (14,16,21,28). Because our results implicated PI(4,5)P 2 as being downstream of RhoA signaling to ENaC, we tested whether RhoA and dominant negative as well as constitutively active mutants of this GTPase influenced ENaC levels in the plasma membrane. Fig. 7A shows a representative Western blot probed with anti-Myc antibody containing whole cell lysate from cells not transfected (negative control) and those overexpressing Myc-tagged ENaC alone and with co-expression of wild type, dominant negative (T19N), and constitutively active (G14V) RhoA. The membrane fractions (from 100 g of total protein) and total cellular pools (from 50 g of total protein) of ENaC are shown for each group. The membrane fraction was isolated by streptavidin precipitation of total cell lysates created from cells that had their plasma membrane proteins labeled with sulfo-NHS-LC-biotin. This approach reliably separates integral plasma membrane from cytosolic proteins as established by counter probing against a cytosolic protein (not shown, but see Ref. 8.). In these experiments, ␣and ␤ENaC migrated as overlying ϳ85-kDa bands and ␥ENaC as an ϳ80-kDa band, which is similar to that observed previously (8,29). As summarized in Fig. 7B, the percentage of ENaC in the plasma membrane in cells expressing the channel alone was 11.5 Ϯ 2.3%. Co-expression with wild type and constitutively active RhoA significantly increased membrane levels to 31.3 Ϯ 4.7 and 32.7 Ϯ 7.4%, respectively. Co-expressing dominant negative RhoA had little effect on the membrane levels of ENaC (7.2 Ϯ 2.9%). These results demonstrate that RhoA increases the membrane levels of ENaC by ϳ3-fold, which is similar to the 2.5-3-fold increase in channel activity observed when ENaC is co-expressed with RhoA. (see Fig. 1B).

DISCUSSION
The current results demonstrate for the first time that ENaC is a final effector of Rho GTPases. This small G protein activated ENaC in a GTP-dependent and C3 exoenzyme-sensitive manner. The effects of RhoA were primarily to increase the membrane levels of ENaC. Activation of ENaC in response to Rho GTPases was sensitive to inhibition of Rho kinase and disruption of PI(4,5)P 2 synthesis and was mimicked by overexpressing PI(4)P 5-K, suggesting that Rho kinase and its known effector PI(4)P 5-K are positioned between the small G protein and the channel with production of PI(4,5)P 2 in response to activation of PI(4)P 5-K increasing membrane levels of ENaC.
In previous work, we showed that Ras GTPases activate ENaC in a GTP-dependent manner (8). However, we also observed activation of ENaC by endogenous GTPases other than Ras. Considering the current findings, an appropriate candidate for an endogenous GTP-dependent protein that activates ENaC is RhoA.
Although GTP is required for RhoA activity, we believe that the fast inhibitory effect of GDP␤S on ENaC activity described in the current work is not directly linked to RhoA activation of the channel. Our rationale is that if RhoA increases ENaC levels in the plasma membrane (see below), GDP␤S is not likely to have a fast inhibitory action on the channel if it impacts ENaC activity by blocking this action of RhoA. We propose that the majority of the fast inhibitory actions of GDP␤S are mediated via Ras or another GTPase the activity of which is directly linked to channel open probability (see below). We further posit that the stimulatory effect of this other GTPase on ENaC open probability then is required to observe augmentation of channel activity by Rho via increasing channel number. More simply put, the channel must be able to open before an effect on channel number can be observed.
The effects of Rho GTPases on ENaC are similar to those described previously for Ras GTPases in the sense that they both increase channel activity (8). However, the mechanisms of activation differ between these two families of GTPases; Ras GTPases increase channel open probability, whereas Rho GTPases, as shown here, increase channel membrane levels. It is interesting that phospholipids appear to serve as signaling intermediates in both instances.
There is good precedence for Rho GTPases via phospholipid signaling influencing cytoskeletal organization to impact insertion and retrieval of integral membrane proteins. Rho GTPases and Rho kinase are recognized activators of PI(4)P 5-K, which is rate-limiting for PI(4,5)P 2 synthesis (11)(12)(13). Rho GTPases signaling via PI(4)P 5-K and production of PI(4,5)P 2 and subsequent actions on actin organization impede endocytosis of the transferring receptor and glucose transporter GLUT4 (28). Actin (re)organization is well documented to influence ENaC activity (30,31), and thus RhoA via phospholipid signaling may feed into this established mechanism modulating ENaC activity. Moreover, Rho GTPases via Rho kinase modulate translocation of aquaporin-2 water channels to the apical plasma membrane of renal principal cells, which also contain ENaC (14,16). It is interesting to speculate that Rho GTPases may play an important role at the cellular level in separating salt and water movement because active forms of these small GTPases retard insertion of aquaporin-2 but, as shown here, promote insertion of ENaC. Rho GTPases, similar to their actions on ENaC, promote insertion of TRPC5 into the plasma membrane (21). This effect on TRPC5 is also dependent on Rho kinase signaling and PI(4)P 5-K activity, again implicating PI(4,5)P 2 as playing a critical role.
Phosopholipids, specifically PI(4,5)P 2 , play critical roles in control of TRP channel gating being necessary and possibly permissive for channel opening (15,32). Similarly, previous reports documenting phospholipid regulation of ENaC showed that PI(4,5)P 2 also impacted channel gating (15). Inclusion of PI(4,5)P 2 in cytosolic solutions prevented channel run-down suggesting that this phospholipid is necessary for channel opening. The current results are not contrary to these earlier observations concerning ENaC but, we believe, complement them by showing that not only is PI(4,5)P 2 required for normal channel gating but that this signaling molecule also plays the parallel role of promoting channel insertion/retention at the membrane level. Although the effect of PI(4,5)P 2 on channel gating appears to be direct, we speculate that the effects of RhoA via PI(4,5)P 2 on membrane levels of ENaC is secondary to actions on cytoskeleton organization and control of vesicle movement. This suggests that ENaC then is similar to TRP channels in that Rho GTPases and PI(4,5)P 2 signaling impact both gating and membrane levels.