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• W2080692942 abstract "Aldosterone-induced serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) contributes to the regulation of the epithelial sodium channel (ENaC), the activity of which is critical for long term blood pressure control. Aldosterone-induced SGK1 is thought to enhance ENaC surface expression by phosphorylating Nedd4-2 and thereby preventing ENaC retrieval and degradation. In outside-out membrane patches of Xenopus laevis oocytes heterologously expressing ENaC, amiloride-sensitive ENaC currents were enhanced by phosphatase inhibitors and were dependent on cytosolic Mg2+. This indicates that a kinase is involved in channel regulation. Indeed, recombinant constitutively active SGK1, included in the pipette solution, caused a sustained 2- to 3-fold increase of ENaC currents. Deletion of the C terminus of αENaC largely reduced the stimulatory effect of SGK1, whereas stimulation by SGK1 did not require the presence of the C termini of the β- or γ-subunits. Replacing the serine residue Ser621 of the SGK1 consensus motif in the C terminus of the α-subunit by an alanine specifically abolished the stimulatory effect of SGK. Our findings indicate that SGK1 can stimulate ENaC activity independently of an inhibition of Nedd4-2-mediated channel retrieval. This defines a novel regulatory pathway likely to be relevant for aldosterone-induced stimulation of ENaC in vivo. Aldosterone-induced serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) contributes to the regulation of the epithelial sodium channel (ENaC), the activity of which is critical for long term blood pressure control. Aldosterone-induced SGK1 is thought to enhance ENaC surface expression by phosphorylating Nedd4-2 and thereby preventing ENaC retrieval and degradation. In outside-out membrane patches of Xenopus laevis oocytes heterologously expressing ENaC, amiloride-sensitive ENaC currents were enhanced by phosphatase inhibitors and were dependent on cytosolic Mg2+. This indicates that a kinase is involved in channel regulation. Indeed, recombinant constitutively active SGK1, included in the pipette solution, caused a sustained 2- to 3-fold increase of ENaC currents. Deletion of the C terminus of αENaC largely reduced the stimulatory effect of SGK1, whereas stimulation by SGK1 did not require the presence of the C termini of the β- or γ-subunits. Replacing the serine residue Ser621 of the SGK1 consensus motif in the C terminus of the α-subunit by an alanine specifically abolished the stimulatory effect of SGK. Our findings indicate that SGK1 can stimulate ENaC activity independently of an inhibition of Nedd4-2-mediated channel retrieval. This defines a novel regulatory pathway likely to be relevant for aldosterone-induced stimulation of ENaC in vivo. The appropriate regulation of the epithelial sodium channel (ENaC) 1The abbreviations used are: ENaC, epithelial amiloride-sensitive sodium channel; ΔIAmi, amiloride-sensitive current; PY motif, PPXY sequence; Nedd4, neuronal precursor cells expressed developmentally down-regulated protein 4; SGK, serum- and glucocorticoid-inducible kinase; SGK1, serum- and glucocorticoid-inducible kinase isoform 1; Po, single channel open probability; NPo, channel activity; wt, wild type; NMDG, N-methyl-d-glucamine; PPI2, protein phosphatase inhibitor type 2; MTSET, (2-(trimethylammonium)ethyl)methanethiosulfonate bromide; GHK, Goldman-Hodgkin-Katz. in the kidney is critically important for the maintenance of body sodium balance and hence for long term regulation of arterial blood pressure (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar). Indeed, two human genetic diseases provide direct evidence that molecular dysfunction of ENaC has severe effects on arterial blood pressure. Loss-of-function mutations of ENaC cause urinary sodium loss, hyperkalemia, and low blood pressure in patients with pseudohypoaldosteronism type 1 (2Chang S.S. Gründer S. Hanukoglu A. Rosler A. Mathew P.M. Hanukoglu I. Schild L. Lu Y. Shimkets R.A. Nelson Williams C. Rossier B.C. Lifton R.P. Nat. Genet. 1996; 12: 248-253Crossref PubMed Scopus (724) Google Scholar). In contrast, gain-of-function mutations of ENaC are found in patients with so-called Liddle's syndrome (pseudohyperaldosteronism) and result in increased renal sodium re-absorption, hypokalemia, and severe arterial hypertension (3Shimkets R.A. Warnock D.G. Bositis C.M. Nelson Williams C. Hansson J.H. Schambelan M. Gill Jr., J.R. Ulick S. Milora R.V. Findling J.W. Canessa C.M. Rossier B.C. Lifton R.P. Cell. 1994; 79: 407-414Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). ENaC is composed of three subunits called α, β, and γ (4Canessa 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). The C termini of the ENaC subunits each contain a proline-rich PPXY (PY) motif, which is believed to be important for interaction with the ubiquitin-protein ligases Nedd4 and Nedd4-2, promoting the ubiquitination, endocytosis, and proteasomal degradation of the channel (5Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 6Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 7Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar, 8Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (601) Google Scholar). The functional importance of the PY motif was recognized in Liddle's syndrome where mutations and/or deletions of the PY motif in β or γ ENaC reduce the endocytic retrieval of ENaC from the membrane (9Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (329) Google Scholar, 10Staub O. Abriel H. Plant P. Ishikawa T. Kanelis V. Saleki R. Horisberger J.D. Schild L. Rotin D. Kidney Int. 2000; 57: 809-815Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). This results in an increase in the number of ENaC channels in the membrane, which in turn is thought to cause hyperabsorption of Na+ and hypertension in patients with Liddle's syndrome (11Firsov 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, 12Kellenberger S. Gautschi I. Rossier B.C. Schild L. J. Clin. Invest. 1998; 101: 2741-2750Crossref PubMed Scopus (149) Google Scholar). The most important hormone to regulate ENaC activity is the mineralocorticoid aldosterone. The effects of aldosterone include transcriptional, translational, and post-translational modifications of ENaC and involve a complex system of aldosterone-induced and/or aldosterone-repressed regulatory proteins (13.Verrey, F., Hummler, E., Schild, L., and Rossier, B. C. (2000) in The Kidney, Physiology & Pathophysiology (Seldin, D. W., Giebisch, G., eds) Third Ed., Chap. 53, pp. 1441–1471Google Scholar). Despite impressive progress in this field of research the molecular mechanisms that mediate the stimulatory effect of aldosterone on ENaC activity remain incompletely understood (14Stockand J.D. Am. J. Physiol. 2002; 282: F559-F576Crossref PubMed Scopus (166) Google Scholar). However, there is a growing body of evidence that the serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) is an important contributing factor in the signal transduction cascade of aldosterone action on epithelial sodium transport (15Pearce D. Cell Physiol. Biochem. 2003; 13: 13-20Crossref PubMed Scopus (142) Google Scholar). In primary cultures of rabbit renal collecting duct cells (16Naray Fejes Toth A. Canessa C. Cleaveland E.S. Aldrich G. Fejes Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar) and in rat distal nephron, aldosterone was shown to stimulate the expression of SGK1 mRNA (17Bhargava A. Fullerton M.J. Myles K. Purdy T.M. Funder J.W. Pearce D. Cole T.J. Endocrinology. 2001; 142: 1587-1594Crossref PubMed Scopus (125) Google Scholar). This effect correlates well with the stimulatory effect of aldosterone on sodium transport and appears to be mediated by the mineralocorticoid receptor (18Shigaev A. Asher C. Latter H. Garty H. Reuveny E. Am. J. Physiol. 2000; 278: F613-F619Crossref PubMed Google Scholar). Immunohistochemical studies have shown that in the renal collecting duct SGK1 is not expressed in intercalated cells but in principal cells consistent with a specific co-expression of SGK1 and ENaC (19Loffing 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). SGK1 mRNA and protein levels are also increased by aldosterone in distal colon (17Bhargava A. Fullerton M.J. Myles K. Purdy T.M. Funder J.W. Pearce D. Cole T.J. Endocrinology. 2001; 142: 1587-1594Crossref PubMed Scopus (125) Google Scholar, 18Shigaev A. Asher C. Latter H. Garty H. Reuveny E. Am. J. Physiol. 2000; 278: F613-F619Crossref PubMed Google Scholar, 20Brennan F.E. Fuller P.J. Mol. Cell. Endocrinol. 2000; 166: 129-136Crossref PubMed Scopus (94) Google Scholar). These findings suggest that SGK1 is a mediator of aldosterone action in classic mineralocorticoid target tissues. SGK1 was the first aldosterone-induced gene shown to upregulate ENaC-mediated sodium transport. This was initially demonstrated by co-expression experiments in Xenopus laevis oocyte (16Naray Fejes Toth A. Canessa C. Cleaveland E.S. Aldrich G. Fejes Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 18Shigaev A. Asher C. Latter H. Garty H. Reuveny E. Am. J. Physiol. 2000; 278: F613-F619Crossref PubMed Google Scholar, 21Chen 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, 22Wagner C.A. Ott M. Klingel K. Beck S. Melzig J. Friedrich B. Wild K.N. Broer S. Moschen I. Albers A. Waldegger S. Tummler B. Egan M.E. Geibel J.P. Kandolf R. Lang F. Cell Physiol. Biochem. 2001; 11: 209-218Crossref PubMed Scopus (100) Google Scholar) and more recently in cultured renal epithelial cells (23Alvarez de la Rosa D. Canessa C.M. Am. J. Physiol. 2003; 284: C404-C414Crossref PubMed Scopus (79) Google Scholar, 24Faletti C.J. Perrotti N. Taylor S.I. Blazer-Yost B.L. Am. J. Physiol. 2002; 282: C494-C500Crossref PubMed Scopus (117) Google Scholar). SGK1 is thought to be an important molecular target that integrates multiple endocrine inputs regulating epithelial sodium transport (24Faletti C.J. Perrotti N. Taylor S.I. Blazer-Yost B.L. Am. J. Physiol. 2002; 282: C494-C500Crossref PubMed Scopus (117) Google Scholar, 25Wang J. Barbry P. Maiyar A.C. Rozansky D.J. Bhargava A. Leong M. Firestone G.L. Pearce D. Am. J. Physiol. 2001; 280: F303-F313Crossref PubMed Google Scholar). The recent development of SGK1 null mice has confirmed that the presence of SGK1 is important for the maintenance of sodium balance, since these animals develop pseudohypoaldosteronism when kept on a low sodium diet (26Wulff P. Vallon V. Huang D.Y. Volkl H. Yu F. Richter K. Jansen M. Schlunz M. Klingel K. Loffing J. Kauselmann G. Bosl M.R. Lang F. Kuhl D. J. Clin. Invest. 2002; 110: 1263-1268Crossref PubMed Scopus (341) Google Scholar). The stimulatory effect of SGK1 occurs in the absence of an increase in ENaC expression levels and appears to be due, at least in part, to increasing the surface expression of ENaC. Recent evidence suggests that the increase in surface expression of ENaC is mediated by PY motif-dependent binding of SGK1 to Nedd4-2 and its subsequent phosphorylation. This has been reported to result in an inhibition of Nedd4-2-mediated ubiquitination, endocytic retrieval, and proteasomal degradation of ENaC, thereby increasing the number of functional ENaC channels in the plasma membrane (27Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Münster C. Chraibi A. Pratt J.H. Horisberger J.-D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (583) Google Scholar, 28Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). However, there is disagreement as to whether the stimulatory effect of SGK1 on ENaC surface expression is primarily mediated by a decrease in removal from or an increase in translocation to the plasma membrane (23Alvarez de la Rosa D. Canessa C.M. Am. J. Physiol. 2003; 284: C404-C414Crossref PubMed Scopus (79) Google Scholar, 29Alvarez de la Rosa D. Zhang P. Naray Fejes Toth A. Fejes Toth G. Canessa C.M. J. Biol. Chem. 1999; 274: 37834-37839Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Finally, in addition to its stimulatory effect on ENaC surface expression, SGK1 may also increase ENaC open probability (30Vuagniaux G. Vallet V. Jaeger N.F. Hummler E. Rossier B.C. J. Gen. Physiol. 2002; 120: 191-201Crossref PubMed Scopus (199) Google Scholar). ENaC phosphorylation by kinases and dephosphorylation by phosphatases (31Becchetti A. Malik B. Yue G. Duchatelle P. Al-Khalili O. Kleyman T.R. Eaton D.C. Am. J. Physiol. 2002; 283: F1030-F1045Crossref PubMed Scopus (24) Google Scholar) has long been thought to contribute to ENaC regulation (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar). Aldosterone, insulin, and protein kinases A and C have been shown to increase in vivo phosphorylation of the C termini of the β- and γ-subunits of ENaC (32Shimkets R.A. Lifton R. Canessa C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3301-3305Crossref PubMed Scopus (162) Google Scholar). Moreover, the C termini of ENaC subunits expressed as glutathione S-transferase fusion proteins were found to be phosphorylated by cytosolic fractions derived from rat colon (33Chigaev A. Lu G. Shi H. Asher C. Xu R. Latter H. Seger R. Garty H. Reuveny E. Am. J. Physiol. 2001; 280: F1030-F1036Crossref PubMed Google Scholar). This phosphorylation is thought to involve at least three different types of kinases, including the extracellular-regulated kinase (34Shi H. Asher C. Chigaev A. Yung Y. Reuveny E. Seger R. Garty H. J. Biol. Chem. 2002; 277: 13539-13547Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and casein kinase 2 (35Shi H. Asher C. Yung Y. Kligman L. Reuveny E. Seger R. Garty H. Eur. J. Biochem. 2002; 269: 4551-4558Crossref PubMed Scopus (40) Google Scholar). In the present report we used outside-out membrane patch recordings to functionally confirm the involvement of kinases and phosphatases in the regulation of ENaC heterologously expressed in X. laevis oocytes. More specifically, we demonstrated a stimulatory effect of recombinant and constitutively active SGK1 on ENaC currents and identified the importance of an SGK consensus motif in the α-subunit for mediating this effect. These results suggest that SGK1 can directly stimulate ENaC activity independently of its effects on Nedd4-2-mediated channel retrieval. Molecular Biology—The full-length cDNAs encoding the three subunits of wild-type (wt) rat ENaC (α-, β-, and γ-ENaC) (4Canessa 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) were in pGEM-HE. Those encoding the truncated rENaC subunits αP646stop, βR564stop, and γF606stop (36Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (362) Google Scholar) and the mutant subunit βS518C (37Kellenberger S. Gautschi I. Schild L. J. Physiol. 2002; 543: 413-424Crossref PubMed Scopus (66) Google Scholar) were in pSD5 and were a gift of Drs. Bernard C. Rossier and Laurent Schild (Lausanne, Switzerland). Linearized plasmids were used as templates for cRNA synthesis using either T7 (wt αβγ-ENaC and truncated γ-ENaC) or SP6 (truncated αβ-ENaC) RNA polymerases (mMessage mMachine, Ambion, Austin, TX). To replace the serine Ser621 in the SGK consensus motif 616RSRYWS621 of rat αENaC by an alanine (αS621A-ENaC) or by aspartate (αS621D-ENaC), site-directed mutagenesis extension overlap PCR was performed using T7 and SP6 as flanking primers. To generate αS621A-ENaC, a mutagenic forward primer with the sequence 5′-CGG AGC CGG TAC TGG GCC CCA GGA CGA GGG GCC-3′ and a reverse primer with the sequence 5′-GGC CCC TCG TCC TGG GGC CCA GTA CCG GCT CCG-3′ were used to introduce a triplet mutation from TCT at nucleotides 1861–1863 to GCC. To generate αS621D-ENaC a mutagenic forward primer with the sequence 5′-CGG AGC CGG TAC TGG GAC CCA GGA CGA GGG GCC-3′ and a reverse primer with the sequence 5′-GGC CCC TCG TCC TGG GTC CCA GTA CCG GCT CCG-3′ were used to introduce a triplet mutation from TCT at nucleotides 1861–1863 to GAC. Mutations were confirmed by sequence analysis. Isolation of Oocytes, Injection of cRNA, and Two-electrode Voltage Clamp Experiments—Isolation and injection of X. laevis oocytes and two-electrode voltage clamp experiments were performed essentially as described previously (38Konstas A.A. Koch J.P. Tucker S.J. Korbmacher C. J. Biol. Chem. 2002; 277: 21346-21351Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Defolliculated stage V–VI oocytes were injected with cRNA using 0.2 or 0.5 ng of cRNA per ENaC subunit. To prevent Na+ overloading (39Konstas A.A. Shearwin-Whyatt L.M. Fotia A.B. Degger B. Riccardi D. Cook D.I. Korbmacher C. Kumar S. J. Biol. Chem. 2002; 277: 29406-29416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) injected oocytes were kept in “low sodium” modified Barth's saline (in mm: 1 NaCl, 40 KCl, 60 N-methyl-d-glucamine (NMDG)-Cl, 0.3 Ca(NO3)2, 0.4 CaCl2, 0.8 MgSO4, 10 HEPES, adjusted to pH 7.4 with HCl, supplemented with 10 units/ml sodium penicillin, and 10μg/ml streptomycin sulfate). Outside-out Macropatch Recordings—After a brief (1–2 min) exposure to hypertonic NMDG-Cl bath solution (in mm: 95 NMDG-Cl, 2 KCl, 1 MgCl2, 1 CaCl2, 200 sucrose, 10 HEPES adjusted to pH 7.4 with Tris) oocytes were stripped of the vitellin membrane using sharpened forceps and transferred to a bath chamber on a Leica DM IRB inverted microscope (Leitz Microsystems UK Ltd., Milton Keynes, UK). A computercontrolled EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany) was used as described previously (38Konstas A.A. Koch J.P. Tucker S.J. Korbmacher C. J. Biol. Chem. 2002; 277: 21346-21351Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) to perform conventional outside-out membrane patch recordings (40Hamill P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15174) Google Scholar). It was usually possible to obtain a second patch from the same oocyte for matched control experiments. Alternatively, a patch from another oocyte of the same batch served as control. For each experimental series oocytes from at least three different batches were used. Patch pipettes were pulled from borosilicate glass capillaries (catalog no. 1155150, inner diameter 0.87 mm, outer diameter 1.5 mm, Hilgenberg, Masfeld, Germany) using a DMZ-Universal puller (Zeitz Instrumente, Munich, Germany) and had a tip diameter of about 5–7 μm after fire polishing to obtain macropatches. Unless stated otherwise pipettes were filled with potassium gluconate pipette solution (in mm: 90 potassium gluconate, 5 NaCl, 2 Mg-ATP, 2 EGTA, and 10 mm HEPES adjusted to pH 7.4 with Tris). A Na2+-free NMDG-Cl bath solution (in mm: 95 NMDG-Cl, 2 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES adjusted to pH 7.4 with Tris) was the standard bath solution at the beginning of each experiment. In NaCl bath solution, NMDG-Cl was replaced by 95 mm NaCl. Downward current deflections correspond to cell membrane inward currents, i.e. movement of positive charge from the extracellular side to the cytoplasmic side. Outside-out patches were routinely held at a holding pipette potential of –70 mV, which was close to the reversal potential of Cl– (ECl = –77.2 mV) and K+ (EK = –79.4 mV) under our experimental conditions. I-V plots were obtained from voltage step protocols, and ΔIAmi values were obtained by subtracting the currents in the presence of amiloride (2 μm) from the corresponding currents prior to addition of amiloride. Single Channel Recordings in Outside-out Patches—Single channel recordings were performed essentially as described previously (38Konstas A.A. Koch J.P. Tucker S.J. Korbmacher C. J. Biol. Chem. 2002; 277: 21346-21351Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 41Letz B. Ackermann A. Canessa C.M. Rossier B.C. Korbmacher C. J. Membr. Biol. 1995; 148: 127-141Crossref PubMed Scopus (36) Google Scholar). Bath and pipette solutions were identical to those used for recordings in outside-out macropatches. The pipette had a tip diameter of about 1 μm after fire polishing. Single channel current data were initially filtered at 250 Hz and sampled at 1 kHz. Data were analyzed using the program “Patch for Windows” written by Dr. Bernd Letz (HEKA Elektronik, Lambrecht/Pfalz, Germany). Using channel traces re-filtered at 15 Hz, channel activity was estimated from binned amplitude histograms as the product NPo, where N is the number of channels and Po is single channel open probability. The program for calculating NPo from integration of the areas under the peaks of amplitude histograms uses the following equation (42Korbmacher C. Volk T. Segal A.S. Boulpaep E.L. Frömter E. J. Membr. Biol. 1995; 146: 29-45Crossref PubMed Scopus (57) Google Scholar): NPo = ∑(nj · ΔIj)/(i · ∑nj), where i refers to the mean single-channel current and where j refers to the jth current amplitude bin, and j ranges from 1 to the total number of bins; nj is the number of events within bin j; ΔIj = Ij – Ic, where Ij is the current of bin j, and Ic is the current at which all channels are closed. Ic was determined in the presence of 2 μm amiloride. Single channel Po was estimated by dividing NPo by the maximal apparent number of channel levels in the current trace analyzed. Solutions and Chemicals—Recombinant constitutively active SGK1(Δ1–60, S422D) was purchased from Biomol GmbH (Hamburg, Germany) as 2-μg vials in 50 μl of stock solution containing as main components 50 mm Tris-HCl, 0.1 mm EGTA, 0.1% 2-mercaptoethanol, 0.15 mm NaCl, and 270 mm sucrose. SGK1 pipette solution was freshly prepared on the day of the experiment by adding 2 μl of the SGK1 stock solution to 1 ml of the potassium-gluconate pipette solution giving a final SGK1 concentration of 80 ng/ml. To preserve SGK1 activity, the pipette solution was supplemented with dithiothreitol (1 mm). For control experiments the pipette solution also contained dithiothreitol (1 mm) and a vehicle control, including the main components of the SGK1 stock solution as indicated above. In addition control experiments were performed using heat-inactivated SGK1 stock solution, which had been incubated at 68 °C for 45 min. Okadaic acid was purchased from Sigma-Aldrich (Taufkirchen, Germany) and was dissolved in Me2SO as a stock solution with a concentration of 0.1 mm. Recombinant protein phosphatase inhibitor type 2 (PPI2) was obtained from Merck Biosciences GmbH (Schwalbach, Germany) as a stock solution containing 1 mg/ml PPI2. Okadaic acid and PPI2 were added to the pipette solution to give final concentrations of 100 nm and 1 μg/ml, respectively. Appropriate amounts of Me2SO or of a buffer corresponding to the main components of the PPI2 stock solution (50 mm Tris-HCl, 1 mm EDTA, 50% glycerol) were added as vehicle controls to the pipette solution in control experiments. The sulfhydryl reagent MTSET ((2-(trimethylammonium)ethyl)methanethiosulfonate bromide) was obtained from Toronto Research Chemicals (Toronto, Canada). Amiloride hydrochloride was purchased from Sigma-Aldrich (Taufkirchen, Germany). ENaC Currents Can Be Recorded in Outside-out Macropatches from X. laevis Oocytes Heterologously Expressing ENaC—In oocytes injected with ENaC cRNA, channel expression was routinely confirmed by using two-electrode voltage clamp measurements, which revealed amiloride (2 μm)-sensitive currents (ΔIAmi) averaging 10.3 ± 1.1 μA at a holding potential of –60 mV (n = 43 in 14 batches of oocytes). From these oocytes it was possible to obtain stable outside-out macropatches in about 50% of attempts. Fig. 1A shows a typical current recording starting about 5 min after patch excision. In the absence of extracellular Na+ in NMDG-Cl bath solution, only a minor inward current component was initially observed. Changing to NaCl bath solution resulted in an immediate increase of the inward current consistent with the occurrence of a current component carried by Na+ influx. After an initial transient peak this Na+ current component relaxed to a slightly lower quasi-steady-state current. The current peak with subsequent relaxation is a well known phenomenon and is most likely due to so-called Na+ self-inhibition by extracellular Na+ thought to reduce the open probability of ENaC through interaction with an extracellular Na+ binding site (43Chraibi A. Horisberger J.D. J. Gen. Physiol. 2002; 120: 133-145Crossref PubMed Scopus (119) Google Scholar). As shown in the current trace in Fig. 1A, application of amiloride, in a concentration of 2 μm known to specifically inhibit ENaC, instantaneously inhibited the Na+ inward current component and the effect was readily reversible upon washout of amiloride. In similar experiments as the one shown in Fig. 1A, ΔIAmi averaged 364 ± 88 pA (n = 22 in 9 batches of oocytes) at a holding potential of –80 mV. Amiloride-sensitive currents could be recorded in all successful outside-out patches from ENaC-expressing oocytes. Moreover, in about 60% of cases it was possible to obtain a second patch with amiloride-sensitive currents from the same oocyte to serve as a direct control experiment. We never detected amiloride (2 μm)-sensitive currents in outside-out patches of non-injected control oocytes (n = 12). Fig. 1B shows I/V plots obtained from voltage step protocols performed in the absence and presence of amiloride during the experiment shown in Fig. 1A. A subtracted I/V plot representing the average ΔIAmi values of similar experiments is shown in Fig. 1C. A Goldman-Hodgkin-Katz (GHK) fit of these data reveals that ΔIAmi is highly Na+ selective as expected for a current mediated by ENaC. ENaC Activity Is Stimulated by Phosphatase Inhibitors and Is Dependent on the Presence of Cytosolic Magnesium—As shown in Fig. 2, we were able to obtain continuous current recordings from outside-out macropatches that were stable for at least 20–30 min. This enabled us to continuously monitor ENaC activity in these patches by repeatedly measuring ΔIAmi. As illustrated in the representative current trace shown in Fig. 2A, ΔIAmi remained largely unchanged throughout the experiment. Maintenance of channel activity may require a balance between channel phosphorylation and dephosphorylation mediated by kinases and phosphatases, respectively, which may be associated with ENaC outside-out macropatches. We therefore tested the effect of phosphatase inhibitors that were included in the pipette solution. ENaC currents increased over time when okadaic acid (100 nm), a known inhibitor of a range of phosphatases, was included in the pipette solution. Similarly, inclusion of PIP2 (1 μg/ml), a more specific inhibitor of phosphatase 1, had a stimulatory effect on ENaC currents as shown in the representative current recording in Fig. 2B. On average, both okadaic acid and PIP2 stimulated ENaC currents by 2- to 3-fold with a similar time course (Fig. 2D). These findings indicate that continuous phosphorylation and dephosphorylation reactions occur in the outside-out macropatches and that the phosphatase inhibitors shift the equilibrium toward phosphorylation resulting in enhanced channel activity. To confirm the kinase dependence of ENaC channel activity we performed additional experiments in which Mg2+ was omitted from the pipette solution, which in addition contained the divalent cation-chelating substance EDTA (10 mm). As shown in the representative current trace in Fig. 2C, ENaC currents decreased to a very low level within 10–15 min under these conditions. Data from similar experiments are summarized in Fig. 2D and indicate that ENaC activity is dependent on the presence of cytosolic Mg2+. This is likely to be due to an Mg2+-dependent kinase that appears to be necessary for maintaining ENaC channel activity. Recombinant SGK1 Stimulates ENaC Currents in Outside-out Patches—To test whether SGK1 can directly affect ENaC activity in outside-out patches, constitutively active recombinant SGK1 (80 ng/ml) was included in the pipette solution. As shown in Fig. 3B, this resulted in a substantial increase of ΔIAmi over time reaching a steady-state level that was on average about 2- to 3-fold higher than that of the initial ΔIAmi (Fig. 3C). In contrast, including heat-inactivated recombinant SGK1 (n = 8, Fig. 3A) or a vehicle control for the SGK1 buffer (n = 22, Fig. 3C) did not stimulate ENaC currents. Data are summarized in Fig. 3C and indicate that recombinant SGK1 applied from the cytosolic side leads to a sustained stimulation of ENaC currents. The average I/V plots shown in Fig. 3D were derived from the set of experiments shown in Fig. 3C with SGK1 in the pipette solution. The I/V plots were constructed using ΔIAmi values that were measu" @default.
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• W2080692942 title "A Novel Pathway of Epithelial Sodium Channel Activation Involves a Serum- and Glucocorticoid-inducible Kinase Consensus Motif in the C Terminus of the Channel's α-Subunit" @default.
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