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Anionic Phospholipids Regulate Native and Expressed Epithelial Sodium Channel (ENaC)

2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês

10.1074/jbc.c100737200

1083-351X

Heping Ma, Sunil Saxena, David G. Warnock,

Neuroendocrine regulation and behavior

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP2 with anti-PIP2 antibody, hydrolysis of PIP2 with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-l-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP2 and PIP3 significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat α-, β-, and γ-ENaC. However, PIP2 and PIP3 did not affect surface expression of ENaC, indicating that PIP2 and PIP3 regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors. Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP2 with anti-PIP2 antibody, hydrolysis of PIP2 with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-l-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP2 and PIP3 significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat α-, β-, and γ-ENaC. However, PIP2 and PIP3 did not affect surface expression of ENaC, indicating that PIP2 and PIP3 regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors. phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 4-phosphate phosphatidylinositol 3,4,5-trisphosphate epithelial sodium channel phospholipase C phosphatidylserine phosphatidylcholine phosphoinositide 3-kinase The phospholipid compositions of the two lipid bilayer leaflets of the plasma membrane are strikingly different. Anionic phospholipids are normally located in the inner leaflet to form a negatively charged surface. However, whether the phospholipid asymmetry affects the function of membrane proteins remains largely unknown. Previous studies have shown that one of the anionic phospholipids, phosphatidylinositol 4,5-bisphosphate (PIP2),1regulates Na+-Ca2+ exchangers and ATP-sensitive potassium (KATP) channels (1Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (563) Google Scholar, 2Hilgemann D.W. Annu. Rev. Physiol. 1997; 59: 193-220Crossref PubMed Scopus (146) Google Scholar). Convincing evidence suggests that PIP2 directly interacts with the proximal COOH terminus of inward-rectifier K+ channels (3Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (771) Google Scholar). Not only PIP2, but also other negatively charged phospholipids such as phosphatidylinositol 4-phosphate (PI-4-P) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), regulate KATPchannels (4Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (487) Google Scholar, 5Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (442) Google Scholar). A model for the regulation of KATPchannels by anionic phospholipids has been proposed, which argues that the negatively charged head group of PIP2, PI-4-P, or PIP3 locks the positively charged carboxyl terminus of KATP channels at a certain position, resulting in the failure of ATP binding to the terminus (6Ashcroft F.M. Science. 1998; 282: 1059-1060Crossref PubMed Google Scholar). This model raises an interesting question: can anionic phospholipids interact with the positively charged cytoplasmic termini of other ion channels? The epithelial sodium channel (ENaC) plays a very important role in regulating total body Na+ homeostasis. Recent studies suggest that PIP2 stimulates ENaC in A6 cells (7Yue G. Eaton D.C. FASEB J. 2000; 14 (abstr.): A340Google Scholar) and that a decrease in PIP2 concentration may account for the inhibition of ENaC by luminal purinergic P2Y receptors (8Ma H.-P. Li L. Zhou Z.-H. Eaton D.C. Warnock D.G. Am. J. Physiol. 2002; 282: F501-F505Crossref Scopus (20) Google Scholar). It is known that ENaC consists of three subunits designated α, β, and γ (9Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar). By examining the first 50 amino acids of the NH2-terminal tails of α-, β-, and γ-ENaC, we found that the NH2-terminal tails of β- and γ-ENaC, but not of α-ENaC, contain significant numbers of positive charges. In fact, the P3geKiKaKiKKnL15 sequence in the γ subunit NH2 terminus is very similar to the pleckstrin homology domain in PLC-δ1 (10Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (618) Google Scholar). We hypothesize that these positive charges might interact with anionic phospholipids of the inner leaflet of the plasma membrane to modulate ENaC activity. Previous studies have shown that deletion of the NH2-terminal tails of β-ENaC (Δ2–49) and γ-ENaC (Δ2–53), but not the NH2-terminal tail of α-ENaC (Δ2–46), dramatically reduces ENaC activity (11Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), suggesting that the positively charged NH2-terminal tails of β- and γ-ENaC play an important role in regulating ENaC activity. It has been shown that positively charged poly-l-lysine partially reversed the effect of PIP2 on KATP channels (12Koster J.C. Sha Q. Nichols C.G. J. Gen. Physiol. 1999; 114: 203-213Crossref PubMed Scopus (82) Google Scholar), indicating that positively charged agents may compete with the positively charged COOH terminus of KATP channels for binding to PIP2. Similarly, the positively charged NH2-terminal tails of β- and γ-ENaC could be physically "locked" by negatively charged phospholipids to the inner surface of the plasma membrane. This putative interaction may account for the role of the NH2-terminal tails of β- and γ-ENaC in regulation ENaC activity (11Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, the present study aims to determine whether anionic phospholipids such as PIP2, PIP3, and PS regulate ENaC activity. A6 distal nephron cells were purchased from American Type Culture Collection (Rockville, MD) at passage 68. The cells were cultured in a plastic flask in a modified NCTC-109 medium (Invitrogen) with 10% fetal bovine serum (Invitrogen) and 1.5 μm aldosterone (Sigma) at 26 °C and 4% CO2. Cells from passages 72–82 were removed from the flasks and plated on permeable supports attached to Snapwell inserts from Corning Costar Co. The permeable supports were coated with rat-tail collagen according to the protocol that is used by Corning Costar Co. The cells were cultured on permeable supports for 10–14 days before patch clamp recordings, as we reported previously (13Ma H. Ling B.N. Am. J. Physiol. 1996; 270: F798-F805PubMed Google Scholar). Most chemicals, including phosphatidylinositol-specific PLC, adenosine 5′-triphosphate, PS, phosphatidylcholine (PC), and poly-l-lysine were obtained from Sigma. PIP2, PIP3, and phosphatase inhibitor mixture were purchased from Calbiochem. Monoclonal anti-PIP2 antibody was from Assay Designs. NaCl bath solution contained (in mm): 100 NaCl, 3.4 KCI, 1 CaCl2, 1 MgCl2, and 10 HEPES, at a pH of 7.4. KCl bath solution contained (in mm): 100 KCI, 5 NaCl, 1 MgCl2, 10 HEPES, and 50 nm Ca2+(after titration with 2 mm EGTA), at a pH of 7.4. All the concentrations throughout this article are shown as the final concentration. Immediately before use, a Snapwell insert was thoroughly washed with NaCl bath solution (see "Chemicals and Solutions") and transferred into the patch chamber mounted in the stage of a Leitz inverted microscope. Using patch clamp techniques, inside-out recordings were established on the apical membrane of A6 cells with polished micropipettes with tip resistance of 2.5–5 megaohms. Under the above culture conditions, a patch seal (seal resistance > 20 gigaohms) was usually formed after releasing positive pressure in the patch pipette or after applying a slightly negative pressure. After establishing the cell-attached mode, only patches containing channel activity without base-line drift were used for experiments. Before forming inside-out patches, NaCl bath solution in the patch chamber was replaced with KCl bath solution. Single-channel currents were obtained with +40-mV applied pipette potential (i.e. V m = −40 mV), filtered at 1 kHz, and recorded on video tapes with a modified Sony PCM video converter (Vetter Instruments). Before digitization with pClamp 8 software (Axon Instruments), single-channel records were low-pass filtered at 100 Hz. The total numbers of functional channels (N) in the patch were estimate by observing the number of peaks detected on the current amplitude histograms. As a measure of channel activity, NP o (number of channels × the open probability, P o) was calculated by using at least 2 min of a single-channel record as we described previously (13Ma H. Ling B.N. Am. J. Physiol. 1996; 270: F798-F805PubMed Google Scholar). Experiments were conducted at 22–23 °C. Oocytes were excised from adult female Xenopus frogs and treated with collagenase. Stage V-VI oocytes were injected with cRNAs for wild-type α-, β-, and γ-ENaC subunits and then were incubated at 18 °C in modified Leibovitz medium. Electrophysiological recordings were performed 24–48 h after the injections using two microelectrodes filled with 3 mm KCl and inserted into the oocyte, as we described previously (14Tucker J.K. Tamba K. Lee Y.J. Shen L.L. Warnock D.G. Oh Y. Am. J. Physiol. 1998; 274: C1081-C1089Crossref PubMed Google Scholar). A voltage step protocol from −120 to +40 mV in increments of 20 mV was used. Between voltage steps the membrane was voltage-clamped at a holding potential of −40 mV. The macroscopic ENaC currents were verified by application of 10 μm amiloride to the bath. The net amiloride-sensitive currents were used to represent ENaC activity. After recording control ENaC currents, the oocytes were taken out of the chamber and injected with 1 μl of H2O, PIP2 (30 μm), or PIP3 (30 μm), respectively. Phosphatase inhibitor mixture (2 μm) was included in each injection. 30 min after these injections, amiloride-sensitive currents were re-measured in these oocytes and compared with the currents before these injections. Using confocal microscopy, the surface expression of ENaC after each experimental manipulation was evaluated; rat β and γ-ENaC subunits were tagged in the extracellular loops with the FLAGTM epitope (DYKDDDDK), which can be recognized by M2 monoclonal antibody, as described previously (15Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (399) Google Scholar). The FLAG-tagged β-, γ-, and α-ENaC cRNAs were injected into Xenopusoocytes. Fluorescent imaging analysis of the expression level by confocal microscopy was carried out, as we described previously (16Saxena S. Quick M.W. Tousson A. Oh Y. Warnock D.G. J. Biol. Chem. 1999; 274: 20812-20817Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The oocytes were then secondarily injected with H2O, PIP2, or PIP3 as described above. The effects of PIP2 and PIP3 on ENaC surface expression were evaluated using the confocal fluorescent imaging methods. A paired t test or analysis of variance for multiple comparisons was used for statistical analysis, as we described previously (13Ma H. Ling B.N. Am. J. Physiol. 1996; 270: F798-F805PubMed Google Scholar). A p value less than 0.05 was considered significant. The G protein-coupled P2Y2 receptor is expressed in renal epithelial cells (17Zambon A.C. Hughes R.J. Meszaros J.G. Wu J.J. Torres B. Brunton L.L. Insel P.A. Am. J. Physiol. 2000; 279: F1045-F1052Crossref PubMed Google Scholar). We have found that ATP inhibits ENaC via a PLC-dependent pathway in A6 cells (8Ma H.-P. Li L. Zhou Z.-H. Eaton D.C. Warnock D.G. Am. J. Physiol. 2002; 282: F501-F505Crossref Scopus (20) Google Scholar). It is well known that activation of PLC hydrolyzes PIP2 to generate IP3 and diacylglycerol and subsequently mobilizes [Ca2+]i. However, recent studies suggest that inhibition of Na+ absorption by the P2Y2receptor occurs independently of an increase in [Ca2+]i (18Lehrmann H. Thomas J. Kim S.J. Jacobi C. Leipziger J. J. Am. Soc. Nephrol. 2002; 13: 10-18Crossref PubMed Google Scholar). Therefore, we hypothesize that a decrease in PIP2 concentration might mediate the P2Y2 receptor-induced inhibition of ENaC. To test this hypothesis, inside-out patch experiments were performed as shown in Fig. 1. We found that ENaC activity in inside-out patches was stable for the initial 4 min. However, a sudden rundown occurred during the period from 4 to 5 min. Interestingly, the channel rundown was clearly prevented when the "cytoplasmic" bath contained 5 μm PIP2. In contrast, application of 100 nm anti-PIP2 antibody to the cytoplasmic bath to chelate endogenous PIP2significantly accelerated the rundown process. Application of exogenous PLC (0.5 unit/ml) to the cytoplasmic bath, which could hydrolyze PIP2, also accelerated the rundown. Furthermore, application of 100 μm ATP in the patch pipette, which presumably activates endogenous PLC, reduced ENaC activity as we recently observed in cell-attached patches (8Ma H.-P. Li L. Zhou Z.-H. Eaton D.C. Warnock D.G. Am. J. Physiol. 2002; 282: F501-F505Crossref Scopus (20) Google Scholar). The initial values ofNP o in the ATP experiments were much lower than the values in other group experiments. We argue that the inhibition of ENaC already occurred before forming the inside-out configuration, because the effect of ATP occurred when the patch pipette was attached to the cell membrane. These data suggest that a decrease in PIP2 concentration at the inner membrane leaflet appears to mediate the inhibition of ENaC by the P2Y2 receptor. It is known that KATP channels are not only regulated by PIP2, but also by PIP3 (4Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (487) Google Scholar). However, the role of PIP3 has been neglected, because the plasma membrane does not contain PIP3 under normal conditions. Nevertheless, PIP3 can be generated by activation of phosphoinositide 3-kinase (PI 3-kinase). Interestingly, recent studies have shown that both aldosterone and insulin enhance Na+transport by activating PI 3-kinase in A6 cells and that inhibition of PI 3-kinase will block their stimulatory effect on ENaC activity (19Blazer-Yost B.L. Paunescu T.G. Helman S.I. Lee K.D. Vlahos C.J. Am. J. Physiol. 1999; 277: C531-C536Crossref PubMed Google Scholar, 20Paunescu T.G. Blazer-Yost B.L. Vlahos C.J. Helman S.I. Am. J. Physiol. 2000; 279: C236-C247Crossref PubMed Google Scholar, 21Record R.D. Froelich L.L. Vlahos C.J. Blazer-Yost B.L. Am. J. Physiol. 1998; 274: E611-E617PubMed Google Scholar). To test whether PIP3 could affect ENaC activity, the inside-out patch configuration was used. Consistent with the results as shown in Fig. 1, ENaC activity in inside-out patches was steady during the initial 4–5 min before a sudden rundown occurred. In contrast, the channel activity was maintained without rundown when the cytoplasmic bath contained 5 μm PIP3 (Fig.2). Because the concentration of PIP3 is elevated in response to aldosterone (19Blazer-Yost B.L. Paunescu T.G. Helman S.I. Lee K.D. Vlahos C.J. Am. J. Physiol. 1999; 277: C531-C536Crossref PubMed Google Scholar), the effect of PIP3 on ENaC activity may account in part for the regulation of ENaC by aldosterone. To test whether other anionic phospholipids can also regulate ENaC activity, inside-out patches were examined when negatively charged PS (20 μm) was applied to the cytoplasmic bath. Similar to the effect of PIP2 and PIP3, anionic PS also prevented ENaC rundown, suggesting that the effect of phospholipids on ENaC activity may be related to their anionic composition. To test whether negative charges are important for the effect of PIP2, PIP3, and PS on ENaC activity, the effect of the positively charged molecule, poly-l-lysine, on ENaC activity was examined. It appears that addition of poly-l-lysine (10 μg/ml) accelerated ENaC rundown. However, neutral PC had no effect on ENaC activity (Fig.2). These data suggest that an increase in PIP3concentration may mediate stimulation of ENaC by corticoid receptors and insulin at the level of interaction of the ENaC complex with the inner leaflet of the plasma membrane. In addition to A6 cells that natively express ENaC when conditioned by aldosterone, the Xenopus oocyte system was also used to test the role of PIP2 and PIP3 in regulating ENaC activity. Using two-electrode voltage clamp techniques exogenously expressed ENaC activity was evaluated with amiloride-sensitive currents following injection of rat α-, β-, and γ-ENaC cRNAs. Amiloride-sensitive currents were compared in the same oocyte before and 30 min after injection of equal volume of H2O (as a control), PIP2 (30 μm), or PIP3 (30 μm), respectively. Amiloride-sensitive currents were not changed in the oocytes injected with H2O (−728 ± 84 nAversus −750 ± 72 nA; n = 7). In contrast, amiloride-sensitive currents were increased, from −797 ± 40 nA to −1091 ± 69 nA (p < 0.001;n = 14) after injection with PIP2 and from −733 ± 31 nA to −1077 ± 73 nA (p < 0.001; n = 10) after injection with PIP3. To further determine whether the increase in amiloride-sensitive currents were related to ENaC trafficking, the density of ENaC on the surface of the plasma membrane was evaluated with confocal surface labeling techniques. The oocytes that expressed FLAGTM-tagged ENaC were injected with equal volume of H2O (as a control), PIP2 (30 μm), or PIP3 (30 μm), respectively. Fluorescent labeling was carried out 30 min after these injections. The data demonstrated that there was no difference in ENaC surface density between each group of oocytes injected with H2O, PIP2, or PIP3 (Fig.3), indicating that PIP2 and PIP3 up-regulate ENaC through a mechanism that appears to be independent of ENaC trafficking as it affects the density of surface expression. Although PIP2, PIP3, and PS failed to enhance ENaC activity in A6 cells, but only maintain the channel activity, it is likely that the stimulatory effect of anionic phospholipids on ENaC activity may be already saturated in A6 cells, which are continuously cultured in the presence of aldosterone. Further experiments will address this hypothesis by using ENaC-expressing renal epithelial cells cultured either in the absence or in the presence of aldosterone. Presumably, without prestimulation by aldosterone, anionic phospholipids will increase the low basal level of ENaC activity in such cells. We (8Ma H.-P. Li L. Zhou Z.-H. Eaton D.C. Warnock D.G. Am. J. Physiol. 2002; 282: F501-F505Crossref Scopus (20) Google Scholar) and others (18Lehrmann H. Thomas J. Kim S.J. Jacobi C. Leipziger J. J. Am. Soc. Nephrol. 2002; 13: 10-18Crossref PubMed Google Scholar) have recently demonstrated that stimulation of the P2Y family, probably the P2Y2 receptor, inhibits ENaC activity in A6 distal nephron cells and amiloride-sensitive Isc in mouse cortical collecting duct principal cells via a pathway that appears to occur independently of an increase in [Ca2+]i. The present study demonstrates that anionic phospholipids activate endogenously expressed ENaC in A6 cells and exogenously expressed ENaC in Xenopusoocytes. Since both chelation of endogenous PIP2 with anti-PIP2 antibody and hydrolysis of endogenous PIP2 with exogenous PLC or extracellular ATP that presumably activates endogenous PLC could reduce ENaC activity (Fig.1), a decrease in PIP2 concentration in the inner leaflet of the plasma membrane may explain inhibition of ENaC by the P2Y2 Receptor. In addition, we have found that PIP3 also regulates ENaC activity (Fig. 2). With the recognition of the role of PIP3 and PI 3-kinase in the responses to aldosterone and insulin on ENaC activity in A6 cells (19Blazer-Yost B.L. Paunescu T.G. Helman S.I. Lee K.D. Vlahos C.J. Am. J. Physiol. 1999; 277: C531-C536Crossref PubMed Google Scholar, 20Paunescu T.G. Blazer-Yost B.L. Vlahos C.J. Helman S.I. Am. J. Physiol. 2000; 279: C236-C247Crossref PubMed Google Scholar, 21Record R.D. Froelich L.L. Vlahos C.J. Blazer-Yost B.L. Am. J. Physiol. 1998; 274: E611-E617PubMed Google Scholar), and the recent recognition of other phosphatidylinositol kinases (22Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), the role of anionic phospholipids in the tonic regulation of ENaC activity at the level of the plasma membrane may well be of general importance. The response to aldosterone is pleotropic and involves sgk kinase as well as changes in PI 3-kinase (23Chen S.Y. Bhargava A. Mastroberardino L. Meijer O.C. Wang J. Buse P. Firestone G.L. Verrey F. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2514-2519Crossref PubMed Scopus (643) Google Scholar, 24Pearce D. Verrey F. Chen S.Y. Mastroberardino L. Meijer O.C. Wang J. Bhargava A. Kidney Int. 2000; 57: 1283-1289Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 25Verrey F. Pearce D. Pfeiffer R. Spindler B. Mastroberardino L. Summa V. Zecevic M. Kidney Int. 2000; 57: 1277-1282Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 26Waldegger S. Barth P. Raber G. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4440-4445Crossref PubMed Scopus (332) Google Scholar). It appears that aldosterone-mediated activation ofsgk kinase rapidly stimulates translocation of ENaC to the apical membrane (27Loffing J. Zecevic M. Feraille E. Kaissling B. Asher C. Rossier B.C. Firestone G.L. Pearce D. Verrey F. Am. J. Physiol. 2001; 280: F675-F682Crossref PubMed Google Scholar), while the experiments described in the legend to Fig. 3, using anionic phospholipids as the putative downstream effectors of the aldosterone response, demonstrate activation of ENaCin situ rather than recruitment or translocation of ENaC complexes to the plasma membrane in the oocyte system. Therefore, an increase in PIP3 concentration in the inner plasma membrane may account in part for the stimulatory effects of aldosterone and insulin on ENaC activity at the level of the inner leaflet of the plasma membrane.