In vitro phosphorylation of COOH termini of the epithelial Na+ channel and its effects on channel activity in Xenopus oocytes

Alexander Chigaev1, Gang Lu1, Haikun Shi1, Carol Asher1, Rong Xu2, Hedva Latter1, Rony Seger3, Haim Garty1, and Eitan Reuveny1

Departments of 1 Biological Chemistry, 2 Immunology, and 3 Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the gamma -subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on beta (S620) was phosphorylated as well. The possible role of gamma T630 and beta S620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na+ and had a high basal Na+ current. Thus mutating gamma T630 or beta S620 may limit the maximal channel activity achieved by a combination of sgk and low Na+.

epithelial sodium channel; serum- and glucocorticoid-dependent kinase; protein kinase; Nedd4; channel phosphorylation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVE NA+ reabsorption in kidney collecting duct, distal colon, lung, and exocrine glands is mediated by an apical Na+-specific channel, termed epithelial Na+ channel (ENaC) (3, 12, 15). The channel is composed of three homologous subunits, denoted alpha , beta , and gamma  (9). They translate 85- to 95-kDa polypeptides that transverse the membrane twice so that both the COOH and NH2 termini are intracellular (8, 24, 28). The crucial role of ENaC in maintaining salt and water balance has been conclusively demonstrated by identifying genetic diseases associated with mutations in ENaC subunits, as well as by the phenotypic analysis of ENaC knockout mice (for review, see Ref. 16).

Regulation of ENaC in the kidney and intestine is done by a number of hormones such as the mineralocorticoid aldosterone, the antidiuretic peptide vasopressin, and insulin (12, 22, 32). Several recent findings strongly suggest that protein phosphorylation is involved in some of these mechanisms. First, it has been demonstrated that the serine/threonine kinase [serum- and glucocorticoid-dependent kinase (sgk)] is induced by aldosterone, and coexpressing sgk with ENaC in Xenopus laevis oocytes results in a several-fold increase in channel activity (10, 21, 26). Second, aldosterone and insulin were found to increase phosphorylation of beta - and gamma -ENaC in transfected Madin-Darby canine kidney (MDCK) cells (27). Finally, the response of A6 cells to both aldosterone and insulin, is inhibited by the phosphoinositide 3-kinase blocker, LY-294002 (7, 23).

A major pathway controlling ENaC's cell surface expression (and activity) involves its interaction with an underlying protein termed Nedd4, which has an ubiquitin ligase activity. The WW domains of Nedd4 bind to the proline-rich PY motifs on the COOH termini of beta - and gamma -ENaC, leading to channel ubiquitination, internalization, and degradation (30, 31). Mutating residues in the PY region of beta  and gamma  impairs this interaction and increases cell surface expression of the channel as well as its open probability (1, 13, 17, 25, 29). This process is sensitive to changes in the intracellular Na+ activity and is thought to play a role in the modulation of transport by cell Na+ (11, 14, 18). Its involvement in other, hormone-induced channel-activating pathways has not been established.

The present study investigates phosphorylation of ENaC subunits and its possible involvement in channel function. The experiments exploited an in vitro assay in which glutathione S-transferase (GST) fusion proteins containing cytoplasmic domains of ENaC subunits are phosphorylated by kinase-enriched cytosolic fractions. By using this assay, we have demonstrated specific phosphorylation of the COOH termini of the three ENaC subunits. Phosphorylation of the gamma -subunit rat ENaC (gamma -rENaC) by a specific cytosolic fraction occurs on T630, a conserved residue located in the immediate vicinity of the PY motif. The analogous residue on the beta -subunit (S620) is phosphorylated too, but in this case 32P incorporates into other residues as well. Mutating either beta S620 or gamma T630 into alanine inhibits the sgk-induced increase in ENaC activity in oocytes preincubated in low-Na+ buffer but not in oocytes preincubated in normal Na+.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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rDNA. The COOH termini of the three rat ENaC subunits (alpha -613-699, beta -557-638, and gamma -564-650) were subcloned in downstream GST in the bacterial expression vector pGEX3X. The desired fragments were amplified by PCR and ligated into the BamH I/EcoR I site of the above vector in frame with GST. Point mutations in pGEX3X were introduced by using a QuikChange site-directed mutagenesis kit. Mutations in full-length ENaC subunits were introduced by generating overlapping PCR products carrying the desired mutation and reamplifying them with the outside primers. All mutations were verified by sequencing. The mouse sgk clone used has been described previously (26). cDNAs coding for the alpha -, beta -, gamma -rENaC in pSPORT-1 were obtained from B. C. Rossier (Institute of Pharmacology, University of Lausanne). The WWII domain of rat Nedd4 cloned into pGEX-2TK was kindly provided by D. Rotin (Hospital for Sick Children, Toronto, ON).

Purification of GST fusion proteins. pGEX plasmids were transformed into the protease-deficient Escherichia coli strain UT5600 (Genetic Stock Center, Yale University, strain 7092). Transformed bacteria were grown for 16-18 h at 37°C with shaking. Cultures were diluted with 400 ml LB-ampicillin and grown for an additional hour, and protein expression was stimulated by the addition of 0.1 mM isopropyl-D-thiogalactopyranoside. Cells were harvested 1 h later, and the fusion proteins were affinity purified on agarose-glutathione beads.

Extraction and fractionation of rat colon cytosol. Rats (Wistar, 9-11 wk old) were used. Mineralocorticoid stimulation of Na+ transport was induced by either in vivo or in vitro incubation with 10-7-10-8 M aldosterone for 2-2.5 h. The in vivo hormonal stimulation was done by subcutaneous implantation of aldosterone-containing osmotic minipumps as described (6, 26). For in vitro incubation, the distal colon was cut longitudinally into two roughly equal portions that were incubated on gelatine sponge rafts with and without 10-7 M aldosterone as described (5, 6).

The colonic tissue fragments were rinsed in buffer A composed of (in mM) 50 glycerophosphate, pH 7.3, 1.5 EGTA, 1.0 EDTA, 1.0 dithiothreitol (DTT), and 0.1 deaerated sodium orthovanadate (2). In experiments testing for direct effects of Na+ on the phosphorylation efficiency, beta -glycerophosphate was replaced by 50 mM of either K-phosphate (low Na+) or Na-phosphate (high Na+). Tissue fragments were suspended in buffer A plus a cocktail of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1.0 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2.0 µg/ml pepstatin A), and the epithelial cell layer was scraped off the connective tissue using a glass slide. Cells were washed by centrifugation and then disrupted on ice by sonication (3 × 5 s). Cell homogenates were centrifuged for 30 min at 30,000 g, and the supernatants were collected and further fractionated. Cytosol extracted from 1-2 colons was applied to a DE-52 column (400 µl resin swelled and prewashed in buffer A). The flow through was collected (fraction 1), and the column was washed with 1 ml buffer A. Three additional cytosolic fractions were eluted by the subsequent additions of 400-µl aliquots of buffer A, supplemented with increasing concentrations of NaCl. The eluting NaCl concentrations were 0.1 M (fraction 2), 0.2 M (fraction 3), and 0.5 M (fraction 4).

The in vitro phosphorylation assay. GST fusion proteins immobilized on agarose-glutathione beads were washed in buffer A. Aliquots of a 10-µl bead slurry were mixed with 50 µl kinase-enriched cytosolic fraction and 47 µl buffer A. The reaction was initiated by the addition of 3 µl ATP mixture composed of 100 µl 1 M MgCl2, 50 µl 0.2 mM ATP, and 15 µl [32P]ATP (10 mCi/ml, 3,000 Ci/mmol). Suspensions were shaken for 30 min at 30°C and then pelleted. Beads were washed several times in HB1B buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 25 mM MgCl2, and 0.05% Triton X-100) and then suspended in SDS gel sample buffer. Samples were boiled for 5 min, resolved on a 12% SDS-PAGE gel, and exposed to X-ray film. In some experiments, recombinant and activated human sgk (Upstate Biotechnology) has been used instead of colonic cytosol. The enzymatic activity of the purified kinase was determined by phosphorylating the synthetic substrate Crosstide (19). The assay mixture contained 25 ng purified enzyme, 1 nmol Crosstide, and 10 µCi [gamma -32P]ATP in 50 µl of the following buffer (in mM): 5 MOPS (pH = 7.2), beta -glycerophosphate, 1 EGTA, 0.2 deaerated sodium orthovanadate, 0.2 dithiothreitol, 15 MgCl2, and 100 µM nonradioactive ATP. Each observation reported below was confirmed by at least three independent measurements using different cytosolic preparations.

Phosphoamino acid analysis. Gel pieces containing 32P-labeled bands were cut out and minced into small fragments in a solution composed of 50 mM ammonium bicarbonate, 0.1% SDS, and 5 mM beta -mercaptoethanol. Proteins were extracted by a 2-h incubation with shaking, and gel pieces were removed by centrifugation. 32P-labeled proteins were precipitated with trichloroacetic acid, pellets were dried, suspended in 6N HCl, and hydrolyzed for 2 h at 110°C. The hydrolyzed amino acids were mixed with phosphoamino acid markers and resolved on a TLC plate. Markers were visualized by spraying the plate with ninhydrin and comparing them with positions of the 32P-labeled spots.

Functional expression in X. laevis oocytes. Stage V-VI oocytes were injected with cRNA mixtures containing 2.5 ng of each ENaC subunit and sgk. Oocytes were maintained for 2-5 days at 17°C in either high-Na+ (96 mM NaCl + 10 µM amiloride) or low-Na+ (10 mM NaCl + 86 mM choline chloride) solutions. To monitor channel activity, oocytes were perfused in high-Na+ medium without amiloride (irrespective of the preincubation buffer), and channel densities were evaluated by recording the amiloride-sensitive (10 µM) current levels as described (26). For in vitro phosphorylation, groups of ~10 oocytes were homogenized in buffer A using a loose-fitting glass homogenizer. They were sedimented at 15,000 g, and the supernatant was collected and used in an in vitro phosphorylation assay.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

To study phosphorylation of the COOH termini of ENaC subunits by cellular kinases, rat colon cytosolic extracts were fractionated on a DE-52 column. Four fractions eluted with 0-0.5 M NaCl were selected for further investigation. They were reacted with the COOH tails of ENaC subunits expressed as GST fusion proteins, in the presence of [gamma -32P]ATP, Mg2+, and phosphatase inhibitors. Figure 1A depicts the incorporation of 32P into the gamma -COOH tail by the four column fractions. The highest activity was obtained with the third fraction (0.2 M NaCl), and it was therefore used for all subsequent experiments. A much smaller signal was obtained with whole cytosol, presumably due to its high-phosphatase activity or the presence of other ATP-hydrolyzing enzymes. Phosphorylation of alpha - and beta -tails was observed as well, whereas GST and the WWII domain of Nedd4 did not incorporate significant amounts of 32P (Fig. 1, B and C). Usually, the GST-ENaC fusion proteins were partly degraded, resulting in multiple bands in Coomassie staining, not all of which are phosphorylated (cf. Fig. 1B). In all experiments, phosphorylation was quantified by phosphoimaging of the band corresponding to the full-length protein, and values were normalized to the Coomassie staining of the same band. Thus results reported below are independent of protein degradation.


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Fig. 1.   In vitro phosphorylation of epithelial sodium channel (ENaC) subunits by kinase-enriched cytosolic fractions. A: phosphorylation of gamma -glutathione S-transferase (GST) by cytosolic fractions obtained as described in MATERIALS AND METHODS. B and C: phosphorylation of various GST fusion proteins by fraction 3, eluted from DE-52 column with 0.2 M NaCl. Autoradiogram and Coomassie staining are depicted.

An amino acid analysis of the phosphorylated species is depicted in Fig. 2A. For gamma -GST, 32P incorporated into threonines, whereas in the case of beta , both serines and threonines were labeled. No phosphorylation of tyrosines could be detected for any of the subunits. Next, we attempted to identify the phosphorylated residues in these subunits by mutating various conserved serines and threonines. For gamma , phosphorylation by the above cytosolic fraction took place only on T630. Mutating this residue into alanine completely abolished 32P incorporation into the fusion protein (Fig. 2B). Mutating the comparable residue on beta (S620A) had a partial effect. In three independent experiments using different cytosolic preparations, the mutation S620A inhibited 56.4 ± 5.1% of the amount of 32P incorporated into beta . Thus this residue is likely to be the phosphoserine detected in the amino acid analysis. However, another, yet unidentified, residue is the major phosphorylation site.


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Fig. 2.   Identification of phosphorylated residues. A: phosphoamino acid analysis of in vitro phosphorylated beta - and gamma -GST. P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. B and C: phosphorylation of gamma -GST (B) and beta -GST (C) carrying various point mutations of conserved serines and threonines. D: phosphorylation of beta , beta S620A, gamma , and gamma T630A after further purification of the above cytosolic fraction on a monoQ column. E: sequence alignment of the relevant region in the COOH tails of beta  and gamma  from different species.

To establish that the same kinase phosphorylates beta S620 and gamma T630, we further purified the above kinase activity on a monoQ column. A particular fraction eluted from the column by ~0.19 M NaCl incorporated 32P into beta S620 with minimal phosphorylation of other residues, and the same fraction also phosphorylated gamma T630 (Fig. 2D). gamma T630 and beta T620 are located in the immediate vicinity of the PY motif and are well conserved in evolution (Fig. 2E). Another well-conserved threonine in this region (i.e., beta T613 or gamma T623) is not phosphorylated by the above cytosolic fraction (Fig. 2, B and C). It should however, be emphasized that identity of the major phosphorylated residue was strongly affected by the cytosol fractionation procedure. A detailed fractionation on a monoQ column did identify a kinase acting on gamma -T623, as well as on other residues (Shi H, Seger R, and Garty H, unpublished observations).

Next, we examined whether pretreatment of rat colon with aldosterone increases the ability of cytosolic kinases to phosphorylate ENaC's COOH tails. Two experimental protocols were used. The first was an in vitro incubation of tissue segments with 10-8-10-7 M aldosterone or diluent for 2.5 h. The second was implantation of aldosterone containing osmotic minipumps and in vivo aldosterone perfusion. Both protocols have been shown before to manifest the hormonal effects on ENaC and sgk (6, 26). The results of these experiments were quite variable. In some experiments, a clear aldosterone-induced stimulation of beta - and gamma -phosphorylation was apparent, but in general this effect was not very reproducible.

It has been recently demonstrated that the serine/threonine kinase sgk is strongly induced by aldosterone and it can activate ENaC on coexpression in X. laevis oocytes (10, 21, 26). Thus sgk may be either the channel-phosphorylating kinase or an "upstream" kinase, whose induction leads to the activation of the channel-phosphorylating kinase. These possibilities were assessed in the experiments summarized in Figs. 3 and 4. First, it was tested whether recombinant-activated sgk can directly phosphorylate GST fusion proteins. No significant incorporation of 32P into any of the channel subunits or the WWII domain of Nedd4 could be detected in these experiments (Fig. 3A). This is in contrast to a strong phosphorylation of gamma  by epithelial cytosol, measured in parallel (last lane in Fig. 3A). The recombinant sgk used was active and effectively phosphorylated the synthetic peptide Crosstide (Fig. 3B). Thus sgk is not likely to be the COOH-tail-phosphorylating kinase monitored in this study.


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Fig. 3.   Phosphorylation of GST fusion proteins by purified recombinant serum and glucocorticoid-regulated kinase (sgk). A: fusion proteins were incubated with (+) and without (-) 100 ng recombinant-activated human sgk and [32P]ATP as described in MATERIALS AND METHODS. For comparison, gamma -GST was also phosphorylated by cytosolic fraction 3 (cyto). B: incorporation of 32P into Crosstide was determined as described in MATERIALS AND METHODS. The enzymatic activity calculated from these measurements corresponds to 580 U/mg sgk. C: Xenopus laevis (X. laevis) oocytes were injected with cRNA mixtures corresponding to ENaC ± sgk. Expression and activation of the kinase was verified by monitoring its effect on the channel activity (see next Fig.). Oocytes were homogenized, and cytosol was extracted and tested for the phosphorylation of gamma -GST. Activity of the gamma -GST phosphorylating kinase was higher in cytosolic fractions from sgk-expressing oocytes by 1.3 ± 0.06-fold (means ± SE of 5 experiments).



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Fig. 4.   sgk-Induced activation of wild-type and mutated ENaC in X. laevis oocytes. X. laevis oocytes were injected with cRNA mixtures corresponding to wild-type or mutated ENaC subunits with (filled bars) or without (open bars) sgk. Matched groups of oocytes were maintained for 3 days in either a high-Na+ (A) or low-Na+ (B) solution. Channel-mediated currents were evaluated from the differences in current-voltage relationships recorded in the presence and absence of 10 µM amiloride. The figure depicts amiloride-sensitive current amplitudes at -100 mV. Means ± SE of 6-10 oocytes in each group are depicted.

The possibility that sgk is an upstream kinase mediating the current phosphorylation was assessed by the following two protocols. In the first, whole cytosol or the kinase-enriched cytosolic fraction was preincubated with purified recombinant sgk and then assayed for phosphorylation of GST-gamma . Such pretreatment was found to have no effect on gamma -COOH-tail phosphorylation (data not shown). A second approach was to compare in vitro phosphorylation of the GST fusion proteins by fractionated cytosol extracted from X. laevis oocytes, which do or do not overexpress sgk. In these experiments oocytes were injected with ENaC ± sgk and assayed for channel activity 3 days later (see below). After an sgk-induced activation of ENaC was established, oocytes were homogenized, and the cytosol was fractionated and assayed for phosphorylation of GST-gamma by the above in vitro protocol. In this case, a significant but very small sgk-induced increase of GST-gamma phosphorylation was detected (Fig. 3C).

Finally, we tested whether mutating gamma T630 or beta S620 into alanine affects channel activity in X. laevis oocytes. It has been previously reported that mutating these residues into either alanine or glutamic acid has no effect on the current amplitude under normal conditions (25). To further explore a possible role of these residues under specific conditions, wild-type and mutated ENaC subunits were expressed with and without sgk. Oocytes were incubated in two different solutions for 48-72 h. In the first, they were maintained in a low-Na+ medium, a treatment that has been shown before to maximalize ENaC's activity by impairing its interaction with Nedd4 (11, 14, 18). In the second, oocytes were maintained in the regular high-Na+ buffer. These oocytes expressed ~3-fold lower Na+ currents and also had lower reversal potentials (6.2 ± 0.7 vs. 30 ± 3.3 mV).1 Hence, the decreased current is likely to reflect downregulation of ENaC by an increased intracellular Na+ (18). In these low-current oocytes the two-point mutations were without effect on the macroscopic current or its activation by sgk (Fig. 4A). A profound effect of these mutations on the response to sgk was seen, however, in the oocytes that were maintained in a low-Na+ buffer (Fig. 4B). In the wild-type channel, the stimulatory actions of low Na+ and sgk were additive, and each of the two stimuli substantially increased the current amplitude. However, in oocytes expressing mutated subunits, sgk could not increase the macroscopic current beyond the level already achieved by preincubation in a low-Na+ solution. A similar phenomenon has been noticed by first recording channel activity in oocytes incubated in high Na+ and then transferring the same oocytes to a low-Na+ medium for 24-48 h (not shown). Table 1 averages data from a large number of oocytes obtained from three to four different batches of oocytes. The results clearly demonstrate the lack of response to sgk by the mutated ENaC in oocytes incubated in low-Na+ but not in high-Na+ solutions. We have also found that truncating the whole COOH tail of beta  or gamma , but not alpha , blunts the response to sgk, and this effect is independent of the incubation conditions (Table 1).

                              
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Table 1.   Channel activity of wild-type and mutated ENaC

Possible interpretations of the above findings are discussed in the next section. One possibility, however, is that different cytosolic Na+ activities in the two groups of oocytes affect the phosphorylation efficiencies of beta - and gamma -ENaC. To evaluate such an option, we have tested for effects of Na+ on the in vitro incorporation of 32P into GST-gamma . In these experiments, the fusion protein was phosphorylated in modified solutions that contained either 50 or <1 mM Na+. The Na+ concentration in the reaction mixture was found to have no effect on the phosphorylation efficiency (data not shown). Thus the different response with high and low Na+ is not likely to reflect a direct effect of Na+ on the phosphorylation reaction.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examined phosphorylation of the COOH termini of ENaC subunits using an in vitro assay. It was motivated by reports indicating a protein kinase-dependent regulation of the channel (7, 10, 20, 21, 27). Indeed, kinase-enriched cytosolic fractions could substantially phosphorylate all three subunits, whereas no incorporation of 32P into GST or the WWII domain of Nedd4 has been seen. Specificity of the response was further established by demonstrating that mutating a single, well-conserved S/T in the immediate vicinity of the PY motif fully blocked incorporation of 32P into the beta - and gamma -fusion proteins. We suggest that these residues are specifically phosphorylated by a cytosolic kinase and such phosphorylation plays a role in channel function. One cannot, however, rule out the possibility that the above mutations indirectly affect incorporation of 32P at another site. It should also be stressed that the relative intensities of different phosphorylation events largely depend on the cytosol fractionation procedure and the presence of different phosphatases in the tested fraction. Thus no 32P incorporation could be seen when the fusion proteins were phosphorylated by whole cytosol. On the other hand, a more detailed fractionation by fast-performance liquid chromatography identified additional phosphorylated residues, not detected using the crude DE-52 fraction (Shi H, Reuveny E, Seger R, and Garty H, unpublished observations).

Several approaches have been used to elucidate cellular roles of the phosphorylations observed. First, we explored whether pretreating the tissue with aldosterone increases the incorporation of 32P into GST-beta or -gamma . This hormone is a major regulator of ENaC activity and has been reported to increase COOH-tail phosphorylation in transfected MDCK cells (27). The results were quite variable and essentially negative. This, however, does not exclude involvement of aldosterone in channel phosphorylation. Cell homogenization and cytosol fractionation should cause a large dilution of small factors and loss of compartmentalization, which enable new protein/protein interactions. These may reverse or blunt an in vivo activation of kinases by aldosterone.

Next, we evaluated the possible involvement of sgk in the phosphorylation of gamma T630 or beta S620. The enzyme did not directly phosphorylate the COOH termini of any of the three ENaC subunits (Fig. 3), nor did it facilitate phosphorylation by the kinase-enriched cytosolic fraction. Some activation could be observed using cytosol extracted from sgk-overexpressing oocytes, but this effect was quite small (Fig. 3C). Again, these results do not exclude an in vivo mediating role of sgk that is blunted during cell homogenization and fractionation.

Finally, we examined effects of the phosphorylated residues on channel activity in X. laevis oocytes. Mutating the phosphorylated residues into alanine had no effect on the basal current or its activation by Na+ withdrawal. They did, however, impair the response to sgk measured in oocytes that were preincubated in low-Na+-containing solution that had high channel activity. A similar inhibition was recorded in oocytes that had a high basal activity due to the truncation of beta  or gamma ; however, this effect was independent of the Na+ activity in the incubation medium (Table 1). This observation suggests a physiological role of gamma T630 and beta S620, but its interpretation is not trivial. The simplest interpretation is that these mutations tend to limit the maximal channel activity in the oocyte membrane. Both sgk and low-external Na+ increase the amiloride-sensitive Na+ currents by elevating cell surface expression of ENaC without increasing the total amount of channel protein (4, 18). Thus one may speculate that mutating gamma T630 and/or beta S620 decreases the size of an sgk-dependent intracellular pool from which channels are recruited to the plasma surface. Such an effect may have no consequence on the cell response to sgk in oocytes expressing low levels of ENaC on the cell surface (i.e., have a large intracellular pool), but it may be inhibitory if the intracellular pool is further depleted by the preincubation in a low-Na+ buffer. Alternatively, the response to sgk may be mediated by a Na+-dependent process(es) that also requires phosphorylation of the above residues. According to these interpretations, phosphorylation of beta S620 and gamma T630 is not directly related to the sgk response. The first possibility also predicts that dissociating ENaC from Nedd4 by truncating beta  and/or gamma  should blunt the response to sgk, independently of external Na+. This indeed was found to be the case (Table 1). This result, however, differs from the one reported in a previous study (4). The reason for the difference between the two sets of data is not clear. It may reflect different expression levels, or the fact that the channels studied in (4) lacked all three COOH-tails, whereas we have mutated one subunit at a time. Another unexpected observation is that incubation in high Na+ lowered the Na+ currents expressed, in spite of the presence of amiloride during the incubation period. This is likely to reflect Na+ loading through amiloride-insensitive pathways as was indeed indicated by the lower reversal potential. Other possibilities, such as a toxic effect of amiloride cannot be ruled out.

In summary, the present study identifies phosphorylated residues in the COOH termini of beta - and gamma -ENaC and provides an initial indication of their physiological role.


    ACKNOWLEDGEMENTS

This study was supported by research grants from the Israel Science Foundation and The Dolfi and Lola Ebner Center for Biomedical Research (to H. Garty and E. Reuveny).


    FOOTNOTES

In both cases currents were recorded in the same high-Na+ buffer.

Address for reprint requests and other correspondence: H. Garty, Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 Israel, (E-mail: h.garty{at}weizmann.ac.il).

1  In both cases, currents were recorded in the same high-Na+ buffer.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 October 2000; accepted in final form 11 February 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 280(6):F1030-F1036
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