Cell Surface Expression of the ROMK (Kir 1.1) Channel Is Regulated by the Aldosterone-induced Kinase, SGK-1, and Protein Kinase A
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m212301200
ISSN1083-351X
AutoresDana Yoo, Bo Young Kim, Cristina Campo, Latreece Nance, Amanda King, Djikolngar Maouyo, Paul A. Welling,
Tópico(s)Hormonal Regulation and Hypertension
ResumoThe Kir1.1 (ROMK) subtypes of inward rectifier K+ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney. The Kir1.1 (ROMK) subtypes of inward rectifier K+ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney. Extracellular potassium homeostasis, maintained by the regulation of renal potassium excretion, is dependent on the activity of weakly inward rectifying "small conductance" potassium channels (SK) 1The abbreviations used are: SK, small conductance potassium channels; PKA, protein kinase A; SGK-1, serum-glucocorticoid-regulated kinase; HRP, horseradish peroxidase; BSA, bovine serum albumin; HA, hemagglutinin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; EGFP, enhanced green fluorescent protein; WT, wild type; MOPS, 4-morpholinepropanesulfonic acid; ENaC, epithelial Na+ channel; NHERF, sodium-hydrogen exchange regulatory factor; RLU, relative light units. that are expressed on the apical membrane of epithelial cells in the distal nephron (1Frindt G. Palmer L.G. Am. J. Physiol. 1989; 256: F143-F151Crossref PubMed Google Scholar, 2Wang W. Schwab A. Giebisch G. Am. J. Physiol. 1990; 259: F494-F502Crossref PubMed Google Scholar). Encoded by the ROMK (Kir 1.1 or KCNJ1) gene (3Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (835) Google Scholar, 4Lu M. Wang T. Yan Q. Yang X. Dong K. Knepper M.A. Wang W. Giebisch G. Shull G.E. Hebert S.C. J. Biol. Chem. 2002; 277: 37881-37887Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), these Kir channels are thought to be the major, but not exclusive (5Frindt G. Palmer L.G. Am. J. Physiol. 1987; 252: F458-F467Crossref PubMed Google Scholar, 6Woda C.B. Bragin A. Kleyman T.R. Satlin L.M. Am. J. Physiol. 2001; 280: F786-F793Crossref PubMed Google Scholar), route for potassium transport into the tubule lumen and constitute a final regulated component of the potassium secretory machinery of the kidney (7Giebisch G. Am. J. Physiol. 1998; 274: F817-F833Crossref PubMed Google Scholar, 8Palmer L.G. Am. J. Physiol. 1999; 277: F821-F825Crossref PubMed Google Scholar). Indeed, aldosterone, vasopressin, and other factors precisely regulate SK activity, controlling potassium excretion in accord with the demands of potassium balance. Because ROMK channels normally exhibit a very high open probability, near unity, physiologic augmentation of channel activity, as controlled by hormones and dietary potassium (9Wang W. Sackin H. Giebisch G. Annu. Rev. Physiol. 1992; 54: 81-96Crossref PubMed Scopus (129) Google Scholar), is achieved largely by regulated changes in the number of active channels on the plasmalemma. Although the precise molecular mechanisms responsible for physiological augmentation of ROMK channel surface density have remained unclear, a growing body of evidence has pointed to an important role of protein kinase A (PKA) phosphorylation. Like the native secretory channel (10Wang W. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1998; 88: 9722-9725Crossref Scopus (141) Google Scholar), ROMK activity is dependent on direct PKA phosphorylation (11McNicholas C.M. Wang W. Ho K. Hebert S.C. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8077-8081Crossref PubMed Scopus (140) Google Scholar, 12Xu Z.C. Yang Y. Hebert S.C. J. Biol. Chem. 1996; 271: 9313-9319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), possibly facilitated by A-kinase-associated proteins (13Ali S. Chen X. Lu M. Xu J.Z. Lerea K.M. Hebert S.C. Wang W.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10274-10278Crossref PubMed Scopus (60) Google Scholar). In fact, activation of ROMK by PKA is thought to underlie the regulation of renal potassium transport by vasopressin (14Cassola A.C. Giebisch G. Wang W. Am. J. Physiol. 1993; 264: F502-F509PubMed Google Scholar). All three PKA phospho-acceptor sites in ROMK, embedded within the cytoplasmic NH2-(Ser-44) and COOH termini (Ser-219 and Ser-313), must be phosphorylated for full channel function (15MacGregor G.G. Xu J.Z. McNicholas C.M. Giebisch G. Hebert S.C. Am. J. Physiol. 1998; 275: F415-FF22PubMed Google Scholar). Interestingly, single channel experiments revealed that the different PKA phospho-acceptors regulate the channel through different mechanisms (15MacGregor G.G. Xu J.Z. McNicholas C.M. Giebisch G. Hebert S.C. Am. J. Physiol. 1998; 275: F415-FF22PubMed Google Scholar). Phosphorylation of the two COOH-terminal sites are required to maintain the channel in a high open probability state (15MacGregor G.G. Xu J.Z. McNicholas C.M. Giebisch G. Hebert S.C. Am. J. Physiol. 1998; 275: F415-FF22PubMed Google Scholar), controlling both pH-dependent gating (16Leipziger J. MacGregor G.G. Cooper G.J. Xu J. Hebert S.C. Giebisch G. Am. J. Physiol. 2001; 279: F919-F926Google Scholar) and phosphatidylinositol 4,5-biphosphate-dependent activation of the channel (17Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (136) Google Scholar). Phosphorylation of the NH2-terminal site, on the other hand, has no effect on channel open probability. Instead, it appears to control the number of active channels on the cell surface (15MacGregor G.G. Xu J.Z. McNicholas C.M. Giebisch G. Hebert S.C. Am. J. Physiol. 1998; 275: F415-FF22PubMed Google Scholar), reminiscent of the way vasopressin affects the activity of the native secretory channel (14Cassola A.C. Giebisch G. Wang W. Am. J. Physiol. 1993; 264: F502-F509PubMed Google Scholar). Just how this is achieved remains a fundamental question in the field. In particular, it is unknown whether phosphorylation of the serine 44 residue simply switches a pre-existing pool of inactive channels on the membrane into an active gating mode or drives cell surface expression by a vesicular trafficking process. Close inspection of the NH2-terminal PKA site in ROMK1 reveals that it also falls within a canonical serum- and glucocorticoid-regulated kinase (SGK1) phosphorylation sequence (recognized by a RXRXX(S/T) (18Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (530) Google Scholar, 19Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (482) Google Scholar)), suggesting that the channel, and serine 44 in particular, might also be a target of SGK1. This observation has potentially important physiological ramifications. SGK1, a member of the PKB/Akt family of serine/threonine kinases (20Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans;2001/RE17)PubMed Google Scholar), is an immediate early aldosterone-induced gene product (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, 22Naray-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) in the renal collecting duct (22Naray-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, 23Bhargava 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, 24Loffing J. Zecevic M. Feraille E. Kaissling B. Asher C. Rossier B.C. Firestone G.L. Pearce D. Verrey F. Am. J. Physiol. 2002; 280: F675-F682Google Scholar), which has been shown recently to regulate epithelial Na+ channel (ENaC) cell surface expression (25Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster 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) and, consequently, act as a key mediator of the early aldosterone effect on sodium transport in the distal nephron. Conceivably, SGK1 might also control the number of functional ROMK channels at the plasmalemma and explain how aldosterone regulates renal potassium secretion. It should be pointed out that an early test of this idea, using a co-expression assay with the Xenopus orthologue of SGK-1 and the mammalian ROMK channel, failed to detect a permissive action of the kinase (26Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar). Nevertheless, it has remained uncertain whether the negative result was a consequence of the xenogeneic nature of the experimental design, or the requirement of A-kinase-associated protein-like proteins, or the high level of basal phosphorylation state of ROMK in oocytes. Interestingly, a recent report that SGK activation of the sodium-hydrogen exchanger-3 is facilitated by the sodium-hydrogen exchange regulatory factor-2 (NHERF-2) (27Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), a PDZ protein found in the collecting duct (28Wade J.B. Welling P.A. Donowitz M. Shenolikar S. Weinman E.J. Am. J. Physiol. 2001; 280: C192-C198Crossref PubMed Google Scholar) that directly interacts with ROMK, 2D. Yoo and P. A. Welling, unpublished observations. provided further incentive to evaluate if mammalian SGK-1 regulates the ROMK channel. The goals of the present study were to determine the mechanism by which phosphorylation of serine 44 in ROMK controls channel activity and to determine whether this residue is a substrate for the aldosterone-induced kinase, SGK-1. Here we show that SGK-1 phosphorylates serine 44 to drive cell surface expression, providing a potential mechanistic explanation for aldosterone-dependent regulation of the secretory potassium channel. Molecular Biology—A hemagglutinin (HA) epitope tag was introduced into the extracellular loop of ROMK by overlap extension PCR at position 113 of ROMK1. Both ends of the epitope were flanked by two glycine residues to enhance accessibility and flexibility of the extracellular HA tag, creating a sequence which reads 111EFGGYPYDVPDYAGGYP. ROMK1 serine 44 was changed to alanine or aspartic acid using Stratagene's QuikChange Site-directed mutagenesis kit. The mouse SGK-1 cDNA, encoding the entire reading frame, was amplified from reverse-transcribed mouse kidney RNA with the PCR by using SGK-1-specific primers (5′-GGGACGATGACCGTCAAAGC-3′, and 5′-AGCACTCAGAGGAAGGAATCCAC-3′). All constructs used for studies in Xenopus oocytes were subcloned between the 5′- and 3′-untranslated region of the Xenopus β-globin gene in the modified pSD64 vector to increase expression efficiency (29Krieg P.A. Melton D.A. Nucleic Acids Res. 1984; 12: 7057-7070Crossref PubMed Scopus (1081) Google Scholar). This vector also contains a polyadenylate sequence in the 3′-untranslated region (dA23dC30). With the exception of EGFP-ROMK, all constructs used for mammalian expression were subcloned into pcDNA 3.1+ (Invitrogen). EGFP was engineered onto the NH2 terminus of HA-ROMK by subcloning the epitope-tagged Kir1.1 in-frame with EGFP in the pEGFP-Cl (Clontech). The sequence of all amplified or modified cDNAs was confirmed by dye termination DNA sequencing (University of Maryland School of Medicine Biopolymer Core). cRNA Synthesis—Complementary RNA was transcribed in vitro in the presence of capping analogue G(5′)ppp(5′) from linearized plasmids containing the cDNA of interest using SP6 RNA polymerase (mMessage Machine, Ambion Inc.). cRNA was purified by phenol/chloroform extraction and precipitated with ammonium acetate/isopropyl alcohol. Yield was quantified spectrophotometrically and confirmed by agarose gel electrophoresis. Oocyte Isolation and Injection—Oocytes from female Xenopus laevis (Xenopus Express, Homosassa, FL) were isolated and maintained using the standard procedures as described previously (30Welling P.A. Am. J. Physiol. 1997; 273: F825-F836PubMed Google Scholar). Briefly, frogs were anesthetized with 0.15% 3-aminobenzoate, and a partial oophorectomy was performed through an abdominal incision. Oocyte aggregates were manually dissected from the ovarian lobes and then incubated in OR-2 medium (82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, and 5 mm HEPES, pH 7.5) containing collagenase (type 3, Worthington) for 2 h at room temperature to remove the follicular layer. After extensive washing with collagenase-free OR-2, oocytes were stored at 19 °C in OR-3 medium (50% Leibovitz's medium, 10 mm HEPES, pH 7.4). 12–24 h later, healthy looking Dumont stage V–VI oocytes were pneumatically injected with 50 nl of diethyl pyrocarbonate-treated water containing 0–1 ng of cRNA and then stored in OR-3 medium at 19 °C for 2–6 days. Electrophysiology—Whole cell currents in Xenopus oocytes were monitored using a two-microelectrode voltage clamp as described previously (30Welling P.A. Am. J. Physiol. 1997; 273: F825-F836PubMed Google Scholar, 31Flagg T.P. Tate M. Merot J. Welling P.A. J. Gen. Physiol. 1999; 114: 685-700Crossref PubMed Scopus (43) Google Scholar). Briefly, oocytes were bathed in a 45 mm K solution (45 mm KCl, 45 mm N-methyl-d-glucamine-Cl, 1 mm MgCl2, 1 mm CaCl2, 5 mm HEPES, pH 7.4). Voltage sensing and current injecting microelectrodes had resistances of 0.5–1.5 megohms when backfilled with 3 m KCl. Once a stable membrane potential was attained, oocytes were clamped to a holding potential of –20 mV, and currents were recorded during 500-ms voltage steps, ranging from –100 to +40 mV in 20-mV increments. Data were collected using an ITC16 analogue to digital, digital to analogue converter (Instrutech Corp.), and filtered at 1 kHz and digitized on line at 2 kHz using Pulse software (HEKA Electronik) for later analysis. ROMK currents are taken as the barium-sensitive inward current (2 mm barium acetate) as we have done before (31Flagg T.P. Tate M. Merot J. Welling P.A. J. Gen. Physiol. 1999; 114: 685-700Crossref PubMed Scopus (43) Google Scholar, 32Flagg T.P. Yoo D. Sciortino C.M. Tate M. Romero M.F. Welling P.A. J. Physiol. (Lond.). 2002; 544: 351-362Crossref Scopus (27) Google Scholar). Values reported in the text are the barium-sensitive inward currents at –100 mV. Cation permeability ratios (PK/PNa) were estimated from the change in reversal potential induced by replacing extracellular potassium with an equivalent concentration of sodium as we have done before (30Welling P.A. Am. J. Physiol. 1997; 273: F825-F836PubMed Google Scholar). Surface Expression—Plasmalemma expression of the external HA-tagged ROMK1 channel was measured in single oocytes following procedures outlined by Zerangue et al. (33Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar) with slight modifications. In these studies, oocytes were washed two times in cold OR-2 medium, fixed with 4% formaldehyde in OR-2 for 15 min at 4 °C, and washed 3 times in OR-2. To block spurious antibody binding, oocytes were then incubated for 1 h at 4 °C in OR-2 containing 1% bovine serum albumin (BSA). Exposed HA epitopes on the surface of intact oocytes were labeled with a rat monoclonal anti-HA antibody (Roche Applied Science, 1 μg/ml, 3F10, 1% BSA, 4 °C overnight), and then oocytes were washed at 4 °C with OR-2 and incubated with HRP-coupled goat anti-rat (The Jackson Laboratories, 1 μg/ml, 1% BSA, 4 °C, 3 h). Cells were washed for 1 h at 4 °C with OR-2 containing 1% BSA then again for 10 min in OR-2 medium without BSA. Individual oocytes were placed in 50 μl of enhanced chemiluminescence substrate (Amersham Biosciences) and incubated for 1 min at room temperature. Luminescence from single oocytes was measured for 10 s in a Sirius luminometer and reported as relative light units per s. Western Blot Analysis—Oocytes were processed following the protocol described by Kamsteeg and Deen (34Kamsteeg E.J. Deen P.M. Biochem. Biophys. Res. Commun. 2001; 282: 683-690Crossref PubMed Scopus (46) Google Scholar) to isolate proteins from total membrane. In brief, oocytes were washed twice in homogenization buffer (80 mm sucrose, 5 mm MgCl2,5mm NaH2PO4,1mm EDTA, 20 mm Tris, pH 7.4) containing a protease inhibitor mixture (5 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml pepstatin A) and then broken by trituration with a 25-gauge syringe. To pellet yolk proteins and nuclei, homogenates were spun twice at low speed (100 × g) for 10 min. Supernatants were then spun at high speed (14,000 × g) for 20 min at 4 °C to collect the total membrane fraction. Pellets were washed once in the homogenization buffer and spun at top speed again for 10 min and then placed in solubilization buffer (4% sodium deoxycholate, 20 mm Tris, pH 8.0, 5 mm EDTA, 10% glycerol, containing the protease inhibitors) and rocked for 2 h at 37 °C. Particulate material was pelleted (14,000 × g for 20 min at 4 °C), and the solubilized proteins in the supernatant were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in Tris-buffered saline with Tween 20 (0.1%) (TBS-T) containing 5% non-fat dry milk for 1 h at room temperature. Rat anti-HA monoclonal antibody (3F10, Roche Applied Science) was diluted in 5% non-fat dry milk to 0.1 μg/ml and incubated at room temperature for 1 h, washed for 15 min in TBS-T, incubated with goat anti-rat antibody coupled to horseradish peroxidase (HRP) (1:5000, 5% non-fat dry milk), and washed extensively for 20 min in TBS-T. Bound antibodies were then revealed using enhanced chemiluminescence reagent (Amersham Biosciences) and fluorography (X-Omat, Eastman Kodak Co.). Fluorographs were assessed by densitometry using NIH Image. Integrated optical density of each band is reported in arbitrary units where the control is considered to be 1 unit. Immunofluorescence of COS-7—Cells, grown on glass coverslips, were transfected with DNA (2 μg total) using Gene Juice (Novagen). After 2 days, cells were washed in ice-cold modified Ringer's solution (144 mm NaCl, 5 mm KCl, 1 mm CaCl2, 5.5 mm CaCl2, 5.5 mm glucose, 1.2 mm NaH2PO4, 5 mm HEPES, pH 7.4) and fixed with 4% formaldehyde in Ringer's solution for 15 min at 4 °C. One group of cells was permeabilized with 0.1% Triton X-100 for 30 min at room temperature and washed three times in the modified Ringer's solution before blocking. Non-permeabilized cells were not treated with Triton. Both groups where blocked with 1% BSA in the modified Ringer's solution for 30 min at room temperature and labeled with primary antibody in 0.1% BSA (rat anti-HA (Roche Applied Science) at 10 ng/ml; mouse anti-PDI 1:100) for 3 h at room temperature and then washed in modified Ringer's solution three times. Next, cells were incubated with secondary antibody conjugated to either Alexa 488 or 568 (goat anti-rat at 1:100; goat anti-mouse 1:100, Molecular Probes) in 0.1% BSA for 1 h at room temperature in the dark. Slides were then washed and mounted onto slides in VectaShield and sealed with nail polish. Cells were visualized using a 410 Zeiss laser-scanning confocal microscope under a 63× oil immersion lens. Images were acquired at a zoom of 2 and a pinhole size of 18 using 16-frame averaging and were processed with Adobe Photoshop. In Vitro Phosphorylation of ROMK by SGK-1—To determine whether ROMK is a substrate of SGK-1 phosphorylation, in vitro phosphorylation assays were conducted using a constitutively active, recombinant form of SGK-1 (Δ1–60, S422D, SGK-1, Upstate Biotechnology Inc.). Either immunopurified ROMK or ROMK synthetic peptides were used as substrates. For the ROMK immunoprecipitation study, COS-7 cells were transfected with the HA epitope-tagged Kir1.1 using Gene Juice (Novagen). 48 h post-transfection, cells were sequentially washed with ice-cold modified Ringer's solution and lysis buffer (150 mm NaCl, 20 mm Tris-Cl, pH 7.4), harvested in cold lysis buffer with protease inhibitor mixture, and resuspended at 5× cell volume with cold lysis buffer supplemented with protease inhibitor mixture and 1% Triton X-100. Cells were passed through a 27½-gauge needle, incubated on ice for 30 min, and then centrifuged at 15,000 × g for 15 min at 4 °C. Lysates containing the soluble proteins were precleared with 100 μl of 50% Sepharose CL-4B slurry for 2 h rotating at 4 °C. Precleared COS lysates were then rotated overnight with 100 μl of 20% protein-G Sepharose slurry with 2 μg of rabbit anti-HA antibody (Upstate Biotechnology, Inc.). Immunoprecipitates were washed 3 times with lysis buffer containing 0.1% Triton. SGK-1 phosphorylation of immunopurified ROMK bound on Sepharose beads was carried out with an active recombinant SGK-1 purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), according to their recommendations as described below. Once the phosphorylation reaction was complete (30 °C, 60 min), immunoprecipitates were washed 3 times in ADBI buffer (20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 5 mm EGTA, 1 mm sodium orthovanadate, 1 mm dithiothreitol). Bound proteins were eluted by incubating at room temperature for 30 min in 20 μl of SDS sample buffer and run on 12% acrylamide gels. Gels were dried using gel drying sheets (Promega) and exposed to autoradiography film (Kodak, X-Omat) for 1–3 h. In parallel immunoprecipitation reactions, eluates were run on 12% acrylamide gels followed by Western blot analysis as described earlier with rat anti-HA antibody (Roche Applied Science). A synthetic peptide corresponding to the wild type ROMK NH2 terminus (residues, 34–48) and a mutant negative control peptide containing a serine to alanine substitution at position 44 were synthesized by University of Maryland Baltimore Biopolymer core facility. In vitro phosphorylation of the peptides by SGK-1 was carried out according to the manufacturer's recommendation. Briefly, reactions (50 μl), containing the peptide (80 μm), SGK-1 (1–25 ng), [γ-32P]ATP (1 μCi/μl), protein kinase C inhibitor (20 μm), and PKA inhibitor (2 μm) in ADBI buffer, were incubated at 30 °C for 60 min. Aliquots of the reaction were transferred onto the center of a P81 filter paper square and then washed 3 times with 0.75% phosphoric acid followed by another 3 washes in acetone. The P81 squares were placed in scintillation fluid, and bound ratio activity was quantified in a liquid scintillation counter. Aliquots of the reaction were also resolved on Tris-Tricine acrylamide gels (16.5%) followed by autoradiography. To determine the apparent Km values for SGK and PKA phosphorylation of the Ser-44 site in ROMK, in vitro phosphorylation reactions were carried out as described above with varying amounts of the wild type ROMK peptide (0–200 μm) over the initial, linear rate of phosphorylation (0–10 min). Constitutively active recombinant SGK-1, as above, and purified bovine heart PKA catalytic subunit (Upstate Biotechnology, Inc.) were used. Parallel phosphorylation reactions, containing identical specific activities of either SGK-1 or PKA, were performed in triplicate and compared. Specific activity was independently determined by measuring rates of phosphate transfer to the optimum phosphorylation motif of each kinase (SGK (RPRAATF); PKA (GRTGRRNSI)). Quantitation of SGK mRNA Expression by Real Time PCR—Mice, maintained on a standard rodent chow, were placed on a high potassium diet containing 10% KCl for 0.5, 1, 2, and 4 days. Kidneys were harvested, and total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's recommendations. Two micrograms of total kidney RNA was reverse-transcribed by using Omniscript (Qiagen). Serum- and glucocorticoid-regulated kinase-1 or actin cDNAs were separately amplified in parallel real time PCRs, containing an aliquot of the reverse-transcribed reaction, 0.2× SYBR Green I (Molecular Probes), and 50 pmol of either serum- and glucocorticoid-regulated kinase-1 (SGK)-specific primers (forward 5′-TGTCTTGGGGCTGTCCTGTATG-3′; reverse 5′-GCTTCTGCTGCTTCCTTCACAC-3′) or actin-specific primers (forward 5′-GGCATTGTTACCAACTGGGACG-3′; reverse 5′-GCTTCTCTTTGATGTCACGCACG-3′) in separate wells using the Bio-Rad iCycler real time PCR detection system. Each sample was amplified in triplicate. Specificity of the amplified product was determined using melting curve analysis and agarose gel electrophoresis. For quantification, real time PCR of serially diluted SGK cDNA was carried out as a positive control and to establish an optimal threshold cycle detection level; threshold cycle values, Ct, of the test samples were then measured from cycle-dependent product amplification curves (Bio-Rad software package iCycler version 3.0a). The change in SGK transcript, reported as the fold induction over the actin control, was calculated using the 2(–ΔΔCt) method (35Livak K.J. Schmittgen T.D. Methods (Orlando). 2001; 25: 402-408Crossref Scopus (127155) Google Scholar). Statistical Analysis—Statistical analysis was performed using GB-Stat™ for Macintosh statistics package (Dynamic Microsystems, Inc). Analysis of variance followed by Fisher's LSD Post Hoc test was performed to verify significance. Values of at least p < 0.05 were considered significant. All data are given as mean ± S.E. Surface Expression Assay—Phosphorylation of the serine 44 residue regulates ROMK either by activating dormant channels already residing on the plasma membrane or by recruiting new channels to the cell surface, two mechanisms that can be distinguished readily by differences in cell surface expression. Accordingly, we incorporated a hemagglutinin epitope tag into the extracellular loop of the ROMK channel so that plasmalemma channel levels could be quantified accurately by antibody binding. When expressed in Xenopus oocytes, HA-tagged ROMK carried weakly inward-rectifying, K+-selective (PK/PNa >15) and barium-sensitive currents identical to those observed for the wild type channel (Fig. 1). To measure relative amounts of surface HA-tagged ROMK, we employed a method that combines enzyme amplification with the sensitivity and linearity of analytical luminometry as originally described by Zerangue et al. (33Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). In this assay, exposed HA epitopes on the surface of intact oocytes were labeled with a monoclonal antib
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