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J Am Soc Nephrol 13:2823-2830, 2002
© 2002 American Society of Nephrology

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

C. Chris Yun*, Monica Palmada{dagger}, Hamdy M. Embark{dagger}, Olga Fedorenko{dagger}, Yuxi Feng{dagger}, Guido Henke{dagger}, Iwan Setiawan{dagger}, Christoph Boehmer{dagger}, Edward J. Weinman{dagger}, Sabrina Sandrasagra§, Christoph Korbmacher§,¶, Philip Cohen||, David Pearce# and Florian Lang{dagger}

*Department of Medicine, Division of Digestive Disease, Emory University, Atlanta, Georgia; {dagger}Department of Physiology I, University of Tübingen, Tübingen, Germany; {ddagger}Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland; §University Laboratory of Physiology, Oxford, UK; Department of Cellular and Molecular Physiology, University of Erlangen-Nürnberg, Germany; ||School of Life Sciences, HRC, Protein Phosphorylation Unit, University of Dundee, Great Britain; #Department of Medicine, Nephrology Division, University of California, San Francisco, California.

Correspondence to: Prof. Dr. Florian Lang, Physiologisches Institut, Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. Phone: 49-7071/2972194; Fax: 49-7071/295618;E-mail: florian.lang{at}uni-tuebingen.de


   Abstract
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 Abstract
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 Materials and Methods
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 Discussion
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ABSTRACT. Mineralocorticoids stimulate Na+ reabsorption and K+ secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K+ channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pKa of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K+ excretion.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The serum and glucocorticoid-dependent kinase 1 (SGK1) was originally cloned from rat mammary tumor cells as glucocorticoid-inducible gene (1) and from human HEPG2 cells as cell volume-regulated gene (2). Subsequent studies revealed the profound stimulation of SGK1 transcription by mineralocorticoids (3–7). Coexpression of SGK1 increases Na+ channel activity in Xenopus oocytes expressing the renal epithelial Na+ channel ENaC (3–5,8–11) by increasing the abundance of ENaC protein within the cell membrane (8,11,12). Thus, hSGK1 is believed to participate in the regulation of renal Na+ excretion by mineralocorticoids.

The electrical driving force for apical Na+ entry through ENaC is maintained by K+ channels (13). The renal outer medullary K+ channel ROMK1 is considered the most important channel to serve this function (14). Mineralocorticoid stimulation of Na+ reabsorption is paralleled by stimulation of K+ secretion (15). In fact, ROMK1 is similarly subject to regulation by aldosterone (16). Moreover, ROMK1 has been demonstrated to be regulated by phosphorylation (17–19). However, no significant effect of SGK1 on ROMK1 has been observed previously in the Xenopus oocyte system (3).

Another potential regulator of ROMK1 is NHERF2 (20), which is colocalized with the K+ channel in the apical membrane of principal cells (21). NHERF2 modulates the targeting and trafficking of proteins into the plasma membrane (22). As SGK1 apparently regulates transport primarily by increasing the abundance of regulated transport proteins in the plasma membrane (8,11), its action on ROMK1 could at least in theory be dependent on the presence of NHERF2.

The present study has been performed to test for a mutual interaction of SGK1 and NHERF2 in the regulation of ROMK1 channel activity.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression in Xenopus laevis Oocytes and Voltage Clamp Analysis
cRNA encoding constitutively active SGK1 (S422DSGK1) (2,23), wild-type ROMK1 (24), and wild-type NHERF2 (20) were synthesized in vitro as described previously (25). Dissection of Xenopus laevis ovaries, collection, and handling of the oocytes have been described in detail elsewhere (25). Oocytes were injected with 5 ng of rat ROMK1, 7.5 ng of human S422DSGK1, and/or 5 ng of human NHERF2 cRNA or H2O. All experiments were performed at room temperature 2 d after injection of the respective cRNAs. In two-electrode voltage clamp experiments, currents were recorded following a step change of the holding potential from -80 mV to -20 mV. The data were filtered at 10 Hz and recorded with MacLab digital to analog converter and software for data acquisition and analysis (AD Instruments, Castle Hill, Australia). The control bath solution (ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4. All substances were added to the solutions at the indicated concentrations. The final solutions were titrated to the pH indicated using HCl or NaOH. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s.

pH Measurements
pH measurements were performed using ion-selective microelectrodes prepared from pulled borosilicate electrodes, which are backfilled with 5% tributylchlorosilane in carbontetrachloride (Fluka Chemicals, Deisenhofen, Germany). To ensure a smooth coating and to remove the solvent, the electrodes were baked at 400 to 450°C for 5 min. A column of H+ cocktail (proton ionophore II-cocktail A, Fluka Chemicals, Deisenhofen, Germany) of approximatley 300 µm in length was filled into the tip of the electrode. The electrode was then backfilled with a solution of 100 mM Na-citrate at pH 6.0. The electrodes were calibrated using solutions with pH 6.0, 7.0, and 8.0. Only electrodes with a linear slope >50 mV/pH unit and stable calibration were used (25). Signals were recorded with an electrometer (WPI model FD223, Sarasota, FL). On the basis of the calibration curve, for the pH-sensitive electrode, the chemical potentials for H+ (EH+) of oocytes were calculated as the difference between the membrane potential measured simultaneously with a 3 M KCl microelectrode and the electrochemical potential of the pH-sensitive electrode (VH+). Where applicable, intracellular pH has been adjusted by variations of extracellular pH in the presence and absence of 3 mM butyrate.

Cell Surface Biotinylation, Western Blotting, and Immunohistochemistry
For cell surface biotinylation of oocytes, 55 to 75 cells of each group were rinsed three times with ice-cold PBS buffer (pH 8.0). The oocytes were then incubated for 30 min at room temperature in 0.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) diluted in PBS buffer. After washing 4 times with ice-cold PBS, the cells were dissolved in lysis buffer containing 50 mM Tris (pH 7.5), 0.5 mM EDTA (pH 8.0), 0.5 mM EGTA, 100 mM NaCl, 1% Triton X-100, 25 µg/ml aprotinine, and 25 µg/ml leupeptin for 30 min on ice. The solubilized oocytes were centrifuged for 15 min at 14000 x g, then the supernatant was incubated with 50 µl of ImmunoPure Immobilized Streptavidin beads at 4°C overnight. The biotin-streptavidin-agarose bead complexes were then washed 4 times with lysis buffer. The final pellets were dissolved in 20 µl of sample buffer and boiled for 5 min for SDS-PAGE.

The biotinylated membrane proteins were separated by 8% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. After blocking with 5% nonfat dry milk in PBS (pH 7.4)/0.15% Tween 20 for 1 h at room temperature, the blots were incubated with the primary rabbit anti-ROMK1 antibody at 4° overnight (dilution 1:250 in PBS/0.15% Tween 20/2.5% non-fat dry milk). After washing, the first antibody was detected by secondary sheep anti-rabbit IgG antibody conjugated with horseradish peroxidase for 1 h at room temperature. Antibody binding was detected with the enhanced chemoluminescence ECL kit (Amersham, Freiburg, Germany) and exposure to x-ray film.

For determination of ROMK1 expression in whole cell lysates, 30 cells of each group were homogenized in lysis buffer containing 50 mM Tris (pH 7.5), 0.5 mM EDTA (pH 8.0), 0.5 mM EGTA, 100 mM NaCl, 1% Triton X-100, and protein inhibitor cocktail (Roche) at the recommended concentrations. Proteins were transferred to nitrocellulose membranes at 100 V for 90 min. For immunoblotting, rabbit anti-ROMK1 antibody (diluted 1:250 in PBS/0.15% Tween 20/5% nonfat dry milk) was used to detect ROMK1. After blocking with 5% nonfat dry milk in PBS/0.15% Tween 20 for 1 h at room temperature, blots were incubated with the primary antibody at 4° overnight. Secondary peroxidase-conjugated sheep anti-rabbit IgG (diluted 1:1000 in PBS/0.15% Tween 20/5% nonfat dry milk) was used for luminescent detection with an enhanced chemoluminescence (ECL) kit (Amersham, Freiburg, Germany).

Oocytes for immunohistochemistry were devitellinized and fixed in Dent solution overnight at -20°C. After washing with PBS, permeabilization and blocking were performed at room temperature for 30 min by incubation in PBS containing 0.2% Triton-100 and 10% normal goat serum. Then oocytes were incubated with rabbit anti-ROMK1 antibody (dilution 1:250) at 4° for 12 h. The secondary Alexa 488 goat anti-rabbit antibody (dilution 1:200, Molecular Probes, The Netherlands) was added at 4°C for 12 h. The embedding procedure was carried out according to the manufacturer’s instructions (Technovit 7100, Heraeus Kulzer, Germany). Embedded oocytes were cut into 5-µm sections and analyzed with a fluorescence microscope.

Detection of Cell Surface Expression by Chemiluminescence
Defolliculated oocytes were first injected with NHERF2 cRNA (5 ng per oocyte) and/or S422DSGK1 cRNA (12 ng per oocyte), and 2 d later with ROMK1-HA cRNA (90 pg per oocyte). Oocytes were prepared for the surface labeling assay as recently described (26,27), with 1 µg/ml primary, rat monoclonal anti-HA antibody (clone 3F10, Boehringer) and 2 µg/ml secondary, peroxidase-conjugated affinity-purified F(ab')2 goat anti-rat IgG antibody (Jackson ImmunoResearch). Individual oocytes were placed in 50 µl of SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce), and chemiluminescence was quantified in a Turner TD-20/20 luminometer by integrating the signal over a period of 15 s. Results are given in relative light units (RLU). Nonexpressing oocytes were not included in the analysis and were defined as those with a surface expression value within one SD of the mean of water-injected oocytes from the same batch.

For parallel electrophysiologic analyses, injected oocytes were kept in modified Barth solution and were studied approximately 16 h after injection with ROMK1-HA cRNA. Oocytes were routinely clamped at a holding potential of -80 mV. The barium-sensitive current ({Delta}IBa2+) was determined as the difference in current between the presence and absence of 1 mM barium in a KCl solution (95 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, adjusted to pH 7.4 with TRIS).

Statistical Analyses
Data are provided as mean ± SEM; n represents the number of oocytes investigated. All experiments were repeated with at least three batches of oocytes; in all repetitions, qualitatively similar data were obtained. All data were tested for significance by using the t test, and only results with P < 0.05 were considered statistically significant.


   Results
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 Abstract
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 Materials and Methods
 Results
Discussion
 References
 
As shown in Figure 1, the injection of cRNA encoding ROMK1 led to the expression of a K+ current of 7.3 ±0.4 µA (n = 35). The current was almost completely inhibited by addition of 10 mM K+ channel blocker Ba2+. No significant increase of the K+ current was observed after coinjection of the constitutively active kinase S422DSGK1 together with ROMK1 (7.0 ± 0.4 µA; n = 35) under conditions where ENaC-mediated Na+ currents were stimulated fivefold (11). Similarly, the coexpression of NHERF2 together with ROMK1 did not significantly enhance the current (8.2 ± 0.3 µA; n = 35). In contrast, the coexpression of S422DSGK1 together with NHERF2 and ROMK1 led to a statistically significant increase of the current (17.5 ± 1.3 µA; n = 35). In oocytes not injected with ROMK1, the K+-current was negligible (0.05 ± 0.01 µA; n = 6) and not significantly increased by injection of SGK1 (0.06 ± 0.01 µA ; n = 6), NHERF2 (0.05 ± 0.01 µA ; n = 6), or both SGK1 and NHERF2 (0.06 ± 0.01 µA; n = 6).



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  		Figure 1. Upregulation of the renal epithelial K+ channel ROMK1 by NHERF2 and SGK1. Xenopus laevis oocytes were injected with cRNA of ROMK1, S422DSGK1 (SGK1), and/or NHERF2. Addition of 10 mM Ba2+ nearly completely inhibited outward K+ current. Only the combined coexpression of S422DSGK1 and NHERF2 increases ROMK1 channel activity. Arithmetic means ± SEM (n = 35). *** significant difference between expression of ROMK1 alone and coexpression of ROMK1 together with S422DSGK1 (SGK1) and NHERF2 (P < 0.001).

 
The stimulation of ROMK1 channel activity was not paralleled by profound alterations of voltage dependence. As shown in Figure 2, the I/V relation was not significantly affected by the combined application of SGK1 and NHERF2. Given the exquisite pH sensitivity of ROMK1, the stimulating effect of SGK1 and NHERF2 could in theory have been due to cytosolic alkalinization or due to alteration of pH sensitivity of the channel. According to ion-sensitive microelectrodes, the cytosolic pH values approached 7.11 ± 0.02 (n = 5) in oocytes expressing ROMK1 alone and 7.11 ± 0.04 (n = 5) in oocytes expressing ROMK1 together with SGK1 and NHERF2. Moreover, ROMK1 retained its pH sensitivity after coexpression of SGK1 and NHERF2. ROMK1 channel activity was almost abolished by cytosolic acidification with butyrate in both oocytes expressing ROMK1 alone and in oocytes coexpressing ROMK1 together with SGK1 and NHERF2 (Figure 3). However, a detailed analysis of pH dependence revealed a small but significant shift of pKa of ROMK1 toward more acidic values upon coexpression of SGK1 and NHERF2 (Figure 4). To explore whether this shift was secondary to the enhanced K+ channel activity, an additional series was performed on oocytes injected with 20 ng ROMK1 alone. As illustrated in Figure 4 and Table 1, the pKa values were not significantly modified by higher ROMK1 channel expression. Even though statistically significant, the small shift of ROMK1-pH sensitivity upon coexpression of SGK1 and NHERF2 was not sufficient to explain the strong activation of the channel.



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  		Figure 2. I/V relationship of ROMK1 expressing oocytes. The current (I) as a function of the potential difference across the cell membrane (V) in oocytes injected with ROMK1 alone (left) and in oocytes injected with ROMK1 together with NHERF2 and S422DSGK1 (SGK1) (right) at both 2 mM K+ (closed symbols) and 20 mM K+ (open symbols). Arithmetic means ± SEM (n = 6).

 


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  		Figure 3. Inhibition of ROMK1 activity by cytosolic acidification. Influence of extracellular pH (pH 7.4 or 6.5) and of intracellular acidification by addition of 3 mM butyrate on ROMK1-induced current in oocytes expressing ROMK1 alone (B and C) or together with S422DSGK1 (SKG1) and NHERF2 (A and D). Upper panels, original tracings; lower panels, I/V curves.

 


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  		Figure 4. Shift of pH sensitivity of ROMK1 by coexpression with S422DSGK1 (SGK1) and NHERF2. Ordinate: normalized inward current of oocytes expressing either ROMK1 alone (R: 5 ng, {blacksquare}; 20 ng, •) or ROMK1 together with NHERF2 and S422DSGK1 (SGK1) (RNS, {blacksquare}). Currents at different values of internal pH (pHi) were normalized to the maximum inward conductance for that oocyte. Abcissa: intracellular pH (pHi), as controlled by variation of extracellular pH (pHo) in the presence of 3 mM butyrate (see insert).

 

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  		Table 1. pH sensitivity of ROMK1 in the absence or presence of S422DSGK1 (SGK1) plus NHERF2
 
Data from a number of reports have suggested that SGK1 stimulates ENaC-mediated Na+ current by augmenting channel abundance in the plasma membrane (8,11,12). To determine the effect of SGK1 and NHERF2 on ROMK1 abundance in the plasma membrane, Western blot and immunohistochemistry were performed on cell membranes from oocytes expressing ROMK1 together with SGK1 and NHERF2 and in oocytes expressing ROMK1 alone. As shown in Figure 5A, SGK1 and NHERF2 did indeed stimulate ROMK1 plasma membrane abundance as determined by densitometry of Western blots from biotinylated membranes. Moreover, determination of cell surface expression by chemiluminescence again disclosed the stimulating effect of SGK1/NHERF2 on ROMK1 cell surface expression (Figure 6). Increased expression of ROMK1 in the plasma membrane was not due to de novo protein synthesis because Western blots from whole cell lysates did not show any difference in ROMK1 expression in oocytes injected with ROMK1, SGK1, and NHERF2 as compared with those injected with ROMK1 alone (Figure 5B).



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  		Figure 5. Increase of ROMK1 abundance in the cell membrane by coexpression of SGK1 and NHERF2. (A) Expression of ROMK1 in the cell membrane. In oocytes expressing S422DSGK1 (SGK1) and NHERF2, the abundance of ROMK1 channel protein at the surface of the oocyte membrane is significantly increased. ROMK1 abundance in oocytes expressing ROMK1 alone (R) and expressing ROMK1 together with S422DSGK1 (SGK1) and NHERF2 (RNS). Left: Western blots of ROMK1 in biotinylated membrane. Ratio of intensity is compared with the intensity obtained in oocytes expressing ROMK1 alone (R). N.I., for not injected oocytes; + significant difference between oocytes injected with ROMK1 and those injected with water; * significant difference between expression of ROMK1 alone and coexpression of ROMK1 together with S422DSGK1 and NHERF2 (RNS). Arithmetic means ± SEM (n = 3). Right: Immunohistochemistry. ROMK1 staining in the cell membrane of oocytes expressing S422DSGK1 and NHERF2 (RNS) is significantly higher than in oocytes expressing ROMK1 alone (R). (B) Expression of ROMK1 in whole cell lysates. In oocytes expressing S422DSGK1 (SGK1) and NHERF2 the abundance of ROMK1 channel protein in whole cell lysates is not modified significantly as compared with those expressing ROMK1 alone. RS stands for oocytes injected with ROMK1 together with S422DSGK1 (SGK1). Ratio of intensity is compared with the intensity obtained in oocytes expressing ROMK1 alone (R). + significant difference between oocytes injected with ROMK1 and those injected with water. Arithmetic means ± SEM (n = 6).

 


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  		Figure 6. Determination of ROMK1 surface expression by chemiluminescence. Surface expression of extracellularly HA-tagged ROMK1 and {Delta}IBa2+ were assessed in parallel in oocytes coexpressing S422DSGK1 + ROMK1-HA and in oocytes from the same batch coexpressing S422DSGK1 + NHERF2 + ROMK1-HA. Water-injected control oocytes were used to determine nonspecific chemiluminescence and endogenous {Delta}IBa2+. Both {Delta}IBa2+ (open bars) and surface expression of ROMK1-HA (filled bars) were significantly increased in S422DSGK1 + NHERF2 + ROMK1-HA oocytes compared with S422DSGK1 + ROMK1-HA control oocytes (* P < 0.05; ** P < 0.01; *** P < 0.001). Numbers above bars represent the number of oocytes studied. Chemiluminescence as a measure of surface expression is given in relative light units per 15 s per oocyte (RLU/15 s per oocyte).

 
In an additional series of experiments, we examined whether ROMK1-HA surface expression was stimulated using a chemiluminescence assay in parallel with {Delta}IBa2+ measurements. Figure 6 summarizes results from one of three similar experiments. These results demonstrate that both surface expression and {Delta}IBa2+ are increased in oocytes expressing SGK1/NHERF2/ROMK1-HA oocytes compared with SGK1/ROMK1-HA control oocytes. Coexpression of NHERF2 increased {Delta}IBa2+ by 232% and ROMK1-HA surface labeling by 227%. We conclude that increased surface expression of ROMK1-HA is sufficient to explain the stimulation of {Delta}IBa2+ in oocytes coexpressing SGK1/NHERF2 and ROMK1-HA.

In principle, SGK1 and NHERF2 could increase plasma membrane ROMK1 expression by increasing channel insertion, decreasing removal, or a combination of the two. To distinguish these possibilities, K+ currents were monitored in oocytes exposed to brefeldin A (5 µM), which blocks cellular secretory mechanisms by inhibiting vesicle formation at the Golgi apparatus, during different time points (0, 4, 8, 12, and 24 h). The treatment did not affect viability of the oocytes, and the cell membrane potential of noninjected oocytes approached -40 ± 7 mV (n = 24) before and -41 ± 6 mV (n = 24) after 24 h of exposure to brefeldin A. The respective values for nontreated cells were -40 ± 4 mV (n = 24) and -42 ± 4 mV (n = 24) before and after 24> h of incubation in brefeldine-free buffer. Incubation of oocytes expressing ROMK1 in brefeldin A (5 µM)–containing solution led to a gradual decrease of channel activity, which was significantly more rapid after 24 h of incubation in oocytes expressing ROMK1 alone as compared with oocytes expressing ROMK1 together with SGK1 and NHERF2 (Figure 7). This finding points to a stabilizing effect of SGK1 plus NHERF2 coexpression on ROMK1 channel protein in the plasma membrane.



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  		Figure 7. Influence of SGK1 and NHERF2 coexpression on ROMK1 stability. Influence of 5 µM brefeldin A on ROMK1 channel activity with or without coexpression of NHERF2 plus S422DSGK1 (SGK1). Coexpression of S422DSGK1 (SGK1) plus NHERF2 blunts the decrease of channel activity in brefeldine-treated oocytes, determined after 24 h of incubation (A) and at the indicated time points (B). Arithmetic means ± SEM (n = 6). * significant difference between expression of ROMK1 alone and coexpression of ROMK1 together with S422DSGK1 (SGK1) and NHERF2.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present experiments indicate that SGK1 is unable to upregulate ROMK1-mediated K+ current in Xenopus oocytes expressing both proteins, consistent with earlier experiments performed on the closely related ROMK2 channel (3). Interestingly, it appears that this lack of ROMK1 regulation is due to the requirement of NHERF2 expression for the SGK1 effect to be manifested. SGK1 has been shown to regulate the epithelial sodium channel (ENaC) in the absence of NHERF2 (11). In fact, SGK1 stimulates ENaC by phosphorylating, thereby inhibiting the ubiquitin protein ligase Nedd4–2 , which interacts with ENaC in a PY motif-dependent manner (28). The ROMK1 sequence does not include a PY motif. On the other hand, ROMK1 has a PDZ binding motif at the COOH terminus, which is thought to be required for binding to NHERF2 (29). The ENaC sequence does not include a PDZ motif, suggesting that NHERF2 cannot directly interact with ENaC.

The stimulatory effect is not due to changes in ROMK1 channel I/V relation and gating properties (Figure 2). In theory, NHERF2/SGK1 could have been effective through alterations of cytosolic pH, as coexpression of SGK1 with NHERF2 was shown to modulate the activity of the Na+/H+ exchanger 3 (NHE3) (29). However, our data showed that the intracellular pH was not significantly altered by the expression of SGK1 and NHERF2, ruling out this possibility.

Instead, the combined action of SGK1 with NHERF2 enhances the abundance of ROMK1 in the plasma membrane, pointing to a stimulating effect on insertion or an inhibitory effect on the retrieval of the channel protein from the plasma membrane. These two possibilities could be discriminated by brefeldine A, a drug interfering with the insertion of membrane proteins (8,30). In the presence of this drug, no further proteins can be inserted into the plasma membrane and the decay of channel activity reflects the retrieval of channel proteins. The results demonstrate that the decay is significantly blunted by the combined expression of SGK1 and NHERF2, suggesting that SGK1 and NHERF2 affect ROMK1 at least in part by inhibition of protein retrieval. NHERF2 has been previously shown to link membrane proteins to cytoskeletal proteins such as ezrin and actin (20,31). It is hence plausible that the membrane expression of ROMK1 is stabilized by its linkage to the cytoskeleton by NHERF2. This may in part decrease the retrieval of ROMK1 or prolong the retention time at the cell membrane surface.

Even though the small shift of pH sensitivity cannot account for the marked enhancement of channel activity, it indicates that coexpression of NHERF2 and SGK1 does not only enhance channel abundance within the cell membrane but has a subtle but significant effect on channel properties. This effect is not due to enhanced protein abundance or channel activity, as the pH sensitivity was identical in oocytes injected with 5 ng or 20 ng of ROMK1 RNA despite the expected large differences in currents. Rather, SGK1 modifies the channel protein itself. In this respect, it may be of interest that the ROMK1 channel protein contains a consensus sequence for SGK1.

The involvement of different proteins in the regulation of ROMK1 increases the plasticity of K+ channel regulation. One requirement for the upregulation of ROMK1 by the SGK1/NHERF2 mechanism is the genomic upregulation of SGK1. This upregulation is accomplished by aldosterone (3–7), cell shrinkage (2), and a wide variety of additional factors (32). Expressed SGK1 requires activation, which can be accomplished by insulin and IGF1 through PI3 kinase and PDK1 (23,33). Thus, SGK1 integrates the signals coming from aldosterone on the one hand and insulin or IGF1 on the other (34). The involvement of NHERF2 adds to the complexity of this system. In particular, observed variability in K+ excretion in response to mineralocorticoids could be influenced by NHERF2 activity or expression levels, hence accounting for the variability in hypokalemia seen in patients with primary aldosteronism (35). A well-described function of NHERF2 has been the regulation of the Na+/H+-exchanger NHE3, pointing to a role of this molecule in the regulation of the acid base balance (29,36). Renal K+ excretion is a function of acid base balance (13), a correlation attributed to the exquisite sensitivity of ROMK1 to cytosolic pH (37–40). In face of the present observations, it is appealing to speculate that NHERF2 participates in the regulation of K+ excretion by acid base balance. In any case, the effects of NHERF2 are not limited to the regulation of NHE3. Most recently, NHERF2 has been shown to direct the signaling of the PTH receptor (41), an observation further illustrating the diversity of NHERF2 functions.


   Acknowledgments
 
We acknowledge the technical assistance of B. Noll and the meticulous preparation of the manuscript by Tanja Loch. This study was supported by the Deutsche Forschungsgemeinschaft, Nr. La 315/4-4 and La 315/5-1, the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research) 01 KS 9602 and the National Institutes of Health Grant DK-44484 and DK-56695, and the funds from the UK Medical Research Council, Diabetes UK and the Royal Society, to Sir P. Cohen.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication April 22, 2002. Accepted for publication August 7, 2002.




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