| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-subunit of the human epithelial sodium channel is modulated by casein kinase 1Division of 1Pulmonary Medicine and 2Protein Core Facility, The Children's Hospital of Philadelphia, and 3Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia; and 4Departments of Medicine and of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 23 April 2007 ; accepted in final form 16 June 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of 
-hENaC in Xenopus laevis oocytes, and the context of this residue in the COOH terminus of -hENaC is important for this effect. Query of a phosphoprotein database suggested that the -T663 residue of hENaC might be a substrate for phosphorylation by casein kinase 1 (CK1). We tested the hypotheses that phosphorylation of -T663-hENaC by CK1 would regulate the increased functional and surface expression of -T663-hENaC vs. -A663-hENaC in oocytes. General inhibition of CK1 with IC261 decreased the functional and surface expression of -T663-hENaC, but not -A663-hENaC. This decrease in -T663-hENaC functional expression resulted from reduced delivery of -T663-hENaC to the oocyte membrane. IC261 also inhibited the functional expression of -T692-mENaC and a chimeric m(1-678)/h(650-669)-T663, m ENaC, but not -A692-mENaC or m(1-678)/h(650-669)-A663, m ENaC. These data suggest that additional residues outside of the -hENaC COOH terminus are important for modulation of -T663-hENaC trafficking by CK1. Overexpression of CK1 did not alter functional expression of -T663-hENaC. In contrast, modest overexpression of CK1
enhanced, whereas higher levels of CK1 overexpression inhibited -T663-hENaC functional expression. CK1 did not phosphorylate the COOH terminus of either -T663-hENaC or -A663-hENaC in vitro. These data suggest that CK1, and perhaps specifically CK1, regulates the intracellular trafficking of the -A663T functional polymorphism of hENaC indirectly by altering the rate of -T663-hENaC biosynthesis and/or delivery to the plasma membrane. phosphorylation; Xenopus laevis oocytes
Epithelial Na+ channels are composed of three structurally related subunits, termed -, -, and -ENaC. These subunits likely assemble as a 2, 1, 1 tetramer (9, 17), although alternate subunit stoichiometries have also been proposed (26). The three subunits share a common topology of two membrane-spanning domains and intracellular NH2 and COOH termini but have limited (
30 to 40%) sequence identity (5, 20, 27).
Changes in ENaC functional expression are associated with alterations in blood pressure (12, 13). Type I pseudohypoaldosteronism, a disorder characterized by volume depletion, hypotension, and hyperkalemia (7, 22), as well as profuse respiratory secretions and increased mucociliary clearance (15), results from mutations that cause loss of ENaC function. In contrast, Liddle's syndrome, a disorder characterized by volume expansion, hypokalemia, and hypertension (25), results from ENaC gain-of-function mutations. Interestingly, patients with Liddle's syndrome have little apparent pulmonary phenotype (2). Some common polymorphisms of human ENaC may segregate with blood pressure (i.e., T594M) (3), suggesting that ENaC polymorphisms which alter functional channel expression may contribute to the development of hypertension in the general population.
A663T is a common polymorphism in COOH terminus of the -subunit of human ENaC (hENaC), and there are conflicting data reported as to whether this polymorphism segregates with blood pressure (1, 30). We previously showed that Xenopus laevis oocytes expressing wild-type T663 hENaC had significantly higher currents than oocytes expressing A663, and that these higher currents were associated with higher levels of cell surface expression of channels (21) due to a decreased rate of channel removal from the plasma membrane (32). This polymorphism is present in the distal COOH terminus of the -subunit, a region that is not well conserved between human and mouse -subunits and may influence interaction with the cystic fibrosis transmembrane conductance regulator (31). Interestingly, we demonstrated that the A692T mutation in mouse ENaC, corresponding to human A663T, was not associated with differences in functional -mENaC expression, whereas replacement of the distal COOH terminus of the mouse -subunit with the distal COOH terminus of the human -subunit restored the functional differences that were observed with the human A663T polymorphism (21). That T663 is potentially modifiable by phosphorylation, and from our previous observations that mutation of T663 to D663 does not alter the functional expression of hENaC in oocytes (21), suggests the hypothesis that phosphorylation of T663 may regulate its increased functional and surface expression in X. laevis oocytes.
A phosphorylation prediction database search suggested that casein kinase 1 (CK1) might phosphorylate T663. The CK1 family is highly conserved throughout eukaryotes and has been linked to numerous cellular processes including vesicular trafficking. The various isoforms have a highly conserved kinase domain. Previous observations suggest that CK1s are constitutively active and that the compartmentalization and/or subcellular localization of the kinase is an important determinant of CK1-substrate interactions (10, 16). Our data suggest that global inhibition of CK1 decreases the functional and surface expression of T663-hENaC. This may result from altered trafficking of T663-hENaC to the oocyte membrane, and the context of the distal COOH terminus of the ENaC -subunit is not the sole determinant of this effect. Overexpression of CK1, but not CK1, also regulates the activity of T663-hENaC, but this regulation may not result from direct phosphorylation of T663 by CK1.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Expression of ENaC and CK1 in oocytes.
-, -, and -hENaC cDNAs were from M. J. Welsh (University of Iowa). Mouse ENaC (mENaC) cDNAs have been described and used by our group previously (17). All mutants and mouse/human chimeras were described previously by our group (21, 31). cDNAs for CK1 and CK1 were purchased from OmniGene Bioproducts (Cambridge, MA).
cRNAs for wild-type and mutant hENaC and mENaC, wild-type hENaC and mENaC, wild-type hENaC and mENaC, and CK1 and CK1 were synthesized from linearized plasmids containing the appropriate cDNAs using appropriate RNA polymerases (T3, T7, or SP6, mMessage mMachine, Ambion, Austin, TX) and stored at –80°C. cRNA concentration was determined spectroscopically. Stage V-VI oocytes were surgically harvested from female X. laevis (NASCO, Fort Atkinson, WI, or Xenopus Express, Plant City, FL) and pretreated with 2 mg/ml collagenase (type IV, Sigma), as previously described (23). Oocytes were injected with 2 ng/subunit of hENaC cRNAs or 0.33 ng/subunit of mENaC cRNAs in 50 nl of H2O. In some experiments, 2–20 ng of CK1 or CK1 cRNA were coinjected with the hENaC cRNAs. After injection, oocytes were incubated at 18°C in modified Barth's saline [MBS; 88 mM NaCl, 1 mM KCl, 2.4 mM Na HCO3, 15 mM HEPES, 0.3 mM Ca (NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, pH 7.2] supplemented with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamicin sulfate. In some experiments, the CK1 inhibitor IC261 was added to the MBS immediately after injection. The animal protocol was approved by the Children's Hospital of Philadelphia Institutional Animal Care and Use Committee.
Two-electrode voltage clamp.
Two-electrode voltage clamp (TEV) was performed 24–48 h after cRNA injection at room temperature using a DigiData 1320 interface and Axon Geneclamp 500B Amplifier (Axon Instruments, Foster City, CA). Data were acquired at 200 Hz and analyses were performed using pClamp 8.0 or 8.1 software (Axon Instruments) on 833-MHz Pentium III or 3-GHz Pentium 4 Personal Computers (Dell Computer, Austin, TX). Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) with a Micropipette Puller (Sutter Instrument, Novato, CA) and had resistances of 0.5–5 M
when filled with 3 M KCl and inserted into the bath solution. Oocytes were maintained in a recording chamber with 1 ml of bath solution and continuously perfused with bath solution at a flow rate of 4–5 ml/min. The bath solution contained 100 mM Na gluconate, 2 mM KCl, 1.8 mM CaCl2, 3 mM BaCl2, 10 mM tetraethylammonium Cl, and 10 mM HEPES, pH 7.4. A series of voltage steps (1 s) from –140 to +60 mV (adjusted for resting membrane potential) in 20-mV increments were performed, and whole cell currents were recorded 750 ms after initiation of the –100-mV voltage step for data analysis. ENaC-mediated current was defined as the difference in whole oocyte current at –100 mV holding potential (adjusted for resting membrane potential) before and after addition of 10 µM amiloride-HCl (Sigma) to the bath solution.
Whole oocyte and cell surface expression.
Surface expression was examined by a cell surface biotinylation assay as we previously described (21, 31). To facilitate detection of biotinylated hENaC subunits, these experiments used a hENaC with a COOH-terminal V5 epitope tag (-V5), also as previously described (11). Briefly, cRNAs for -V5 hENaC were coinjected into X. laevis oocytes. After 48 h, oocytes were mechanically stripped of their vitelline membranes in hypertonic media (300 mM sucrose in MBS without penicillin, streptomycin, and gentamicin; MBSnoAbx). Oocytes were then washed sequentially with MBSnoAbx, 10 mM triethylamine in MBSnoAbx, and surface proteins were labeled with 1.5 mg/ml sulfo-NHS-Biotin (Pierce) in triethylamine/MBSnoAbx for 30 min on ice. The biotinylation reaction was quenched with 5 mM glycine in MBS (4 separate 5-min incubations on ice). Oocytes (10 per group) were subsequently washed with MBS, lysed in 0.15 M NaCl, 0.01 M Tris·HCl, pH 8.0, 0.01 M EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 1.0 mM phenylmethanesulfonyl fluoride, 0.1 mM N--p-tosyl-L-lysine chloromethyl ketone, 0.1 mM L-1-tosylamide-2-phenylethyl-chloromethyl ketone, and 2 µg/ml aprotinin for 1 h at 4°C, and centrifuged at 13,000 g for 15 min at 4°C. Biotinylated proteins were precipitated with streptavidin-agarose (Pierce) and subjected to SDS-PAGE. Biotinylated -subunits were detected on immunoblots probed with an anti-V5 antibody. Densitometry was performed using an AlphaImager 2200 system (AlphaInnotech, San Leandro, CA).
Whole oocyte expression of -V5-hENaC or CK1 isoforms was assessed by immunoblot of whole oocyte lysate prepared using the lysis buffer and procedure described above. Antibodies to the CK1 and isoforms were purchased from Santa Cruz Biotechnology.
As a control for the integrity of the plasma membrane of the stripped oocytes, we assessed the recovery of biotinylated GAPDH in concurrent experiments. Biotinylated GAPDH was not recovered by streptavidin precipitation as detected by immunoblot despite it being readily detected by whole oocyte immunoblot, suggesting that the oocytes were not leaky after mechanical stripping of the vitelline membrane.
In vitro phosphorylation.
C-terminal gluthatione S-transferase (GST) fusion proteins containing the COOH-terminal 20 amino acids of T663- and A663-hENaC were as previously described (32). GST, GST-T663, or GST-A663 were expressed in Escherichia coli BL21, immobilized on glutathione-sepharose 4B beads (Amersham), and subject to in vitro phosphorylation with a COOH terminally truncated rat CK1 (New England BioLabs), and -[32P]-ATP (10 µCi, 3,000 Ci/mmol, duPont New England Nuclear) according to the manufacturer's protocol. Bound GST or GST fusion proteins were eluted by boiling in SDS-PAGE sample buffer, resolved by SDS-PAGE, and stained with Coomassie blue. Phosphorylation was detected by fluorography. As a positive control for these assays, phosphorylation of a CK1 peptide substrate (Promega) was performed in parallel according to the manufacturer's protocol.
Mass spectroscopic analysis of GST-fusion protein phosphorylation.
In vitro phosphorylation of GST, GST-T663, or GST-A663 with CK1 was performed as described above, except that nonradioactive ATP was used. The GST or GST fusion proteins were resolved by SDS-PAGE, and SPRYO Ruby-stained bands were excised from the gel cut into 1-mm3 cubes using modifications of a published protocol (24). Briefly, gels were destained with 50% acetonitrile/200 mM ammonium bicarbonate, reduced with 20 mM Tris(2-carboxyethyl)phosphine (TCEP; Pierce), and alkylated with 45 mM iodoacetamide. Gels were then washed with 25 mM ammonium bicarbonate followed by 50% acetonitrile/25 mM ammonium bicarbonate. The gel pieces were then dried in a speed vac. Trypsin (20 ng/µl; 50 mM ammonium bicarbonate) was added to the gels until the gel was fully swelled and hydrated. Proteolysis was allowed to proceed at 37°C overnight. Following this, peptides were extracted with 20 mM ammonium bicarbonate, followed by 50% acetonitrile/5% formic acid (FA). Samples were frozen at –80°C until analysis.
Peptide digests were loaded directly onto a C18 capillary column (75 µm x 100 mm; New Objective Proteoprep 2) isocratically in 2% acetonitrile/0.1% FA at a flow rate of 1 µl/min for liquid chromatography/liquid chromatography/mass spectroscopy (LCLCMS) analysis using an Eksigent two-dimensional liquid chromatography (LC) system. A linear gradient was then initiated at a flow rate of 300 nl/min (3–40% buffer B over 42 min; 40–100% buffer B over 3 min; then 5 min at 100% buffer B). Buffer A was 0.1% FA and buffer B was 80% acetonitrile/0.1% FA. Mass spectrometry (MS) was performed on a Thermo-Finnegan LTQ mass spectrometer in a data-dependent fashion as peptides were eluted off of the capillary column. A top 5 method was performed in which one survey scan was followed by MS/MS analysis of the five most intense ions. MS thresholds were set to 1,500 and the MSn (mass spectrometry to the nth power) was set to 500. A mass range of 800–2,500 was implemented for all runs. A repeat count of three was selected such that after three MS/MS repeats this ion was placed onto an exclusion list for 0.5 min. An exclusion window was set to 0.5 Da below the target m/z and 1.5 Da above. MS/MS experiments were performed with an isolation width of 2, and collision energy of 35, and activation Q = 0.25, and an activation time of 30. Peptides that showed a neutral loss of 98, 49.5, or 33.2 Da were subjected for a further round of sequencing (MS/MS/MS or MS3) (4).
Raw sequest datafiles were searched against the species-specific component of the Swissprot database using both Sequest (33) (Thermo) and MASCOT (19) (Matrix Science). Two missed cleavages were allowed. A fixed modification of carbamidimethylation for cysteine and variable modification for methionine oxidation were used. A parent mass window of 1.2 and a fragment tolerance of 0.6 Da were utilized for all ion trap-based searches. For the vacuum matrix-assisted laser desorption/ionization (vMALDI) analyses, the charge state was held to +1, whereas a charge state of +2 and +3 was established for all nanospray analyses. Peptide spectra were only accepted, after Sequest analysis, using the following criteria: Xcorr > 1.5 (z = 1), 2 (z = 2), 2.5 (z = 3); 
Cn > 0.1 and a continuous ion series of at least 5 y-ions and b ions. Additionally, the five major peaks in the MS/MS spectrum needed to be indentified for the peptide to be accepted. Only proteins with a MASCOT score above 60 were accepted. Following the initial search, data were analyzed in detail using the program Scaffold (Proteome Software). Only proteins whose peptide probabilities were above 95% and protein probabilities above 80% were accepted (18). Any protein with a one peptide match was manually inspected and only accepted if a contiguous y and/or b ion series was greater than or equal to five residues.
Assessment of hENaC delivery to and removal from the oocyte plasma membrane.
The rate of delivery of hENaC to the oocyte membrane was assessed as previously described (32), making use of the observations that ENaC mutants where a cysteine was introduced into the "amiloride binding site" (-hENaC G536C) can be irreversibly blocked by treatment with [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET). The time-dependent recovery of benzamil-sensitive whole cell currents reflects delivery of unmodified channels to the cell surface. Benzamil (100 µM) is used for these studies as channels containing amiloride binding site mutations are relatively amiloride insensitive (6, 28). Oocytes were injected with T663-G536C hENaC as described above and subsequently incubated in the absence or presence of 20 µM IC261. Twenty-four to thirty-six hours after injection, whole oocyte currents were determined by TEV before application of MTSET, after two applications of MTSET (1 mM, 5 min each), and every 5 min for 25 min after removal of MTSET and washing of the oocyte. Benzamil (100 µM) was then added, and the remaining whole oocyte current that was insenstitive to benzamil inhibition was determined by TEV. We then calculated the benzamil-sensitive current at a given point by determining the difference between the whole oocyte current at that point and the whole oocyte current remaining after addition of benzamil.
To assess the rate of removal of hENaC from the oocyte membrane, oocytes were injected with cRNAs as described above. Twenty-four to thirty-six hours after injection, amiloride-sensitive current was determined by TEV before (t = 0) and after 2, 4, and 6 h of incubation with Brefeldin A (5 µM). Brefeldin A was used to block delivery of new channels to the oocyte membrane. Amiloride-sensitive currents were expressed relative to the initial amiloride-sensitive current (t = 0), and pseudo first-order rate constants for decline of amiloride-sensitive current were determined for each individual oocyte, as well as the mean and SE (SigmaPlot 2000).
Statistical analyses.
Whole cell amiloride-sensitive current data are expressed relative to that of wild-type ENaC. To decrease the influence of batch-to-batch variability in ENaC expression, data were normalized by the mean amiloride-sensitive current for the control condition (usually T663hENaC) within a batch of oocytes before combining data of multiple independent batches for statistical analysis. These data are presented as means ± SE, and P values were determined by a two-tailed t-test or ANOVA as appropriate. When a Poisson distribution, rather than a Gaussian distribution, best described these combined data, P values were determined by a two-tailed t-test or ANOVA after a square root transformation to better approximate a Gaussain distribution (34). A P value 
0.05 was considered significant. Other data that were normally distributed are expressed as means ± SE with P values determined by a two-tailed t-test or paired t-test as appropriate. All statistical data analyses were performed with SigmaStat version 2.03.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-subunit of the hENaC affects functional and surface expression of -hENaC in X. laevis oocytes. A663 exhibited significantly lower whole cell currents and surface expression and an enhanced rate of removal from the oocyte plasma membrane when compared with T663 (21, 32). This polymorphism is located at the COOH terminus of -hENaC in a region that is poorly conserved across species (Fig. 1). We also demonstrated that the context of this residue in the COOH terminus of -hENaC is important for this effect, as a homologus change in mENaC, A692T, does not alter functional and surface expression of mENaC. However, replacement of the COOH-terminal 21 residues of -mENaC (679-699) with those of -hENaC (650-669) restores the effect (21). Based on these and other supporting observations published by our group (21), we hypothesized that phosphorylation of -T663 may regulate the functional and surface expression of -T663-hENaC. Query of PhosphoBase v2.0 (www.cbs.dtu.dk/databases/PhosphoBase) suggested that the -T663 residue might be a substrate for phosphorylation by CK1. Interestingly, -T692-mENaC was not predicted to be a substrate of CK1 in a similar query. This is consistent with our previous observations that the context of this polymorphism is important for its functional effects (21), and suggested the hypothesis that CK1 might selectively regulate T663 vs. A663-hENaC in the context of the COOH-terminal 20 residues of -hENaC.
|
-T663- and -A663-hENaC in oocytes. Oocytes were injected with -T663 or -A663 hENaC and then incubated either with or without 20 µM IC261, a nonisotype-selective CK1 inhibitor. As shown in Fig. 2A, IC261 decreased ENaC functional expression in oocytes injected with -T663 hENaC but did not alter the functional expression of -A663 hENaC in oocytes. This pattern of -T663- and -A663-hENaC functional expression after exposure to IC261 corresponded to changes in surface expression of these hENaCs in response to IC261 as detected in cell surface biotinylation experiments. In contrast, whole oocyte expression of -V5-hENaC was unaltered by IC261 for both -T663- and -A663-hENaC (Fig. 2B, densitometry of the surface biotinylation experiments is shown in Fig. 2C). These data are thus consistent with CK1 selectively regulating the trafficking of -T663 hENaC in oocytes.
|
-hENaC are necessary determinants of the differential functional and surface expression we observed. Therefore, if a member of CK1 family is "the" kinase that causes the differential functional and surface expression, then we predict that IC261 would not influence the functional expression of -A692T-mENaC, as the context of the T is not "correct." In contrast, we predict that IC261 and CK1 inhibition would alter the functional expression of the chimeric m(1-678)/h(650-669)-A663T, m ENaC, as the context of the T in this construct is "correct" (21). Experiments testing these predictions are shown in Fig. 3. IC261 does not inhibit -A692-mENaC functional expression but, in contrast to our prediction, does inhibit the functional expression of -T692-mENaC (Fig. 3A). These data are inconsistent with the notion that the context of -T692 (homologous to -T663 in the human) is critical for inhibition of CK1 by IC261 to have functional effects. Figure 3B suggests that, as predicted, inhibition of CK1 with IC261 decreases the functional expression of the m(1-678)/h(650-669)-T663, m ENaC chimera, and does not effect the m(1-678)/h(650-669)-A663, m ENaC chimera. These data therefore suggest that CK1 can selectively regulate the trafficking of the -A663T-hENaC polymorphism but that the -T663 reside itself appears sufficient for these effects. The appropriate context of this residue in the COOH terminus of -hENaC is not necessary to observe the effect of CK1 inhibition.
|
A663T polymorphism increases the functional and surface expression of hENaC in oocytes by decreasing the rate at which channels are removed from the plasma membrane (32). We therefore assessed whether the decreased functional and surface expression of -T663-hENaC due to CK1 inhibition was a result of more rapid removal of -T663-hENaC from the oocyte membrane. Figure 4A demonstrates the relative amiloride-senstive currents present in the absence or presence of IC261 before assessing the rate of channel removal and is consistent with our data of Fig. 2A. In the presence of Brefeldin A to block the delivery of new -T663-hENaC channels to the oocyte membrane, the apparent first-order rate constant for loss of -T663-hENaC functional expression in the absence (k = –0.27 ± 0.02 h–1, n = 13) or presence (k = –0.27 ± 0.03 h–1, n = 13, P = not significant) of IC261 was not significantly different (Fig. 4B). These data suggest that CK1 does not influence the retrieval of the A663T-hENaC polymorphism from the plasma membrane.
|
T663-hENaC in the presence of IC261 was due to a decreased rate of delivery of hENaC to the membrane. To assess the rate of delivery, we introduced a cysteine residue into the amiloride binding site of the -hENaC subunit (G536C). This mutant allows for irreversible block of the hENaC channel after treatment with MTSET (28). The rate of recovery of benzamil-sensitive current [with benzamil being used because the mutant channels are relatively insensitive to amiloride (6)] is then a direct measure of the rate of hENaC delivery to the oocyte membrane. These data are shown in Fig. 5. Again, consistent with our results of Figs. 2A and 4A, there was reduced -T663-G536C-hENaC functional expression in the presence of IC261 before assessing channel delivery (Fig. 5A). After irreversible blockade of the surface channels with MTSET, there was a significantly reduced rate of recovery of benzamil-sensitive current over 5 min in the presence (0.10 ± 0.02, n = 12) vs. absence (0.17 ± 0.03, n = 12, P = 0.042) of IC261. These data suggest that CK1 primarily influences the biosynthesis of and/or rate at which the A663T polymorphism is delivered to the oocyte membrane and not the rate at which it is retrieved from the membrane. This contrasts with the mechanism by which the -A663T polymorphism alters hENaC functional and surface expression, where our previous data suggest that the rate of retrieval is altered and the rate of delivery is similar. These data are therefore also inconsistent with CK1 being the sole mediator of the differential functional and surface expression of the -A663T polymorphism of hENaC.
|
-A663T-hENaC functional polymorphism might be selectively regulated by overexpression of specific isoforms of CK1. We focused on the and isoforms of CK1, as these isoforms are suggested to localize with the endoplasmic reticulum, Golgi and/or Trans Golgi Network, and transport vesicular structures (10, 16). Figure 6 demonstrates that CK1 and CK1 were present in immunoblots of oocytes injected with hENaC alone. Coinjection of increasing amounts (2, 10, or 30 ng) of cRNA encoding CK1 or CK1 with hENaC cRNA did lead to a modest increase in the whole oocyte expression of CK1 or CK1. However, as shown in Fig. 6B, overexpression of CK1 did not alter the functional expression of -T663-hENaC. In contrast, Fig. 6D demonstrates that coinjection of a modest amount (2 ng) of CK1 cRNA caused a 40% increase in -T663-hENaC functional expression (P < 0.05). Interestingly, coinjection of a large amount (30 ng) of CK1 cRNA caused a 40% decrease in -T663-hENaC functional expression (P < 0.05). These data suggest that CK1, at appropriate levels of overexpression, can specifically increase the functional expression of -T663-hENaC. We suspect that the decrease in -T663-hENaC functional expression observed with coinjection of large amounts of CK1 represents secondary effects on cellular homeostasis resulting from overexpression of this specific constitutively active kinase. We feel that this result is not due to a theoretical competition for oocyte translational machinery, as coinjection of a similarly large amount of CK1 cRNA does not result in a decrease in -T663-hENaC functional expression.
|
does not directly phosphorylate the COOH terminus of -hENaC.
We next examined whether CK1 selectively phosphorylates the -T663 residue of hENaC. To facilitate these experiments, we utilized our previously constructed GST fusion proteins containing the COOH-terminal 20 residues of -T663- and -A663-hENaC. As shown in Fig. 7, GST, GST--T663, and GST--A663 were all phosphorylated in vitro by CK1. Parallel positive control experiments performed under the same reaction conditions also demonstrated robust phosphorylation of a CK1 peptide substrate (data not shown). These data suggested that GST itself was likely a substrate for CK1.
|
-T663, we performed mass spectroscopic sequence analysis of GST, GST--T663, and GST--A663 that were phosphorylated in vitro by CK1. SPYRO Ruby-stained bands of in vitro phosphorylated GST, GST--T663, and GST--A663 were excised and digested as described in MATERIALS AND METHODS. Samples were analyzed by liquid chromatography followed by tandem ion-trap mass spectrometry. We then used a data-dependent MS/MS/MS analysis in which parent ions that show a predominant loss of a PO43– group are selectively reported and resequenced for a third round of MS-based sequencing. This analysis suggested that all of the CK1-mediated phosphorylation occurred within GST; there was no phosphorylation within the COOH-terminal 20 amino acids of either the GST--T663 or the GST--A663 fusion proteins by CK1. These data therefore suggest that overexpression of CK1 selectively regulates the trafficking of -T663-hENaC to the oocyte membrane by an indirect mechanism rather than by direct phosphorylation of the -T663 residue. |
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-A663T, was associated with altered functional and surface expression in X. laevis oocytes. Specifically, the -T663-hENaC had higher functional and surface expression than the -A663-hENaC and that the context of the COOH-terminal 20 amino acids of -hENaC was critical for this effect (21). We also recently demonstrated that the increased functional and surface expression of the -T663-hENaC resulted from a decreased rate at which the channel was retrieved from the oocyte surface.
To assess the molecular basis underlying the functional effect of this polymorphism, and based on our previous observations (21), we previously established four criteria by which we determined whether an experimental intervention might selectively modulate functional and surface expression of this polymorphic hENaC in the context of the COOH-terminal 20 amino acids of -hENaC (32). 1) The intervention should congruently alter functional and surface expression of either -T663- or -A663-hENaC, but not both. 2) The intervention should not influence the functional expression of -T692- or -A692-mENaC, as the -A692T mutation in mENaC, which is homologous to -A663T in hENaC, does not alter mENaC functional expression in oocytes (21). 3) Replacement of the COOH-terminal 21 amino acids of -mENaC with the COOH-terminal 20 amino acids of -hENaC should resurrect the effect of the -A663T polympophism on the ENaC chimera, and this effect should be the same as that observed for hENaC. Satisfying this third criterion demonstrates that the effect is specific for the context of the COOH-terminal 20 amino acids of -hENaC. 4) If phosphorylation of -T663-hENaC is responsible for the increase in channel activity, activation of the appropriate kinase should selectivity increase the activity of -T663-hENaC.
We recently employed these criteria in testing the hypothesis that PKC, and specifically PKC , might regulate the increased functional and surface expression of T663- vs. -A663-hENaC via the context of the COOH-terminal 20 amino acids of -hENaC (32). Our data suggested that PKC specifically regulated the functional and surface expression of -T663-hENaC in the context of the COOH-terminal 20 amino acids of -hENaC. However, in contrast to our hypothesis, PKC specifically decreased the functional and surface expression of -T663-hENaC and did not alter the rate at which -T663-hENaC was retrieved from the plasma membrane (32). We have now tested a similar hypothesis for CK1, which was also identified as a kinase that could potentially phosphorylate -T663 in hENaC via a phosphorylation predication database.
Global, tonic inhibition of CK1 with IC261 decreased the functional and surface expression of T663-hENaC without influencing A663-hENaC, satisfying the first criterion. Inhibition of CK1 with chronic IC261 exposure also satisfied the third criterion, as the inhibitory effect of IC261 on the m(1-678)/h(650-669)-T663 m ENaC chimera was similar to its effect on T663-hENaC. The CK1 isoform also satisfied the fourth criterion, in that modest overexpression of this isoform resulted in enhanced functional expression of -T663-hENaC. However, the effect of modulation of CK1 activity by inhibition with IC261 did not satisfy the second criterion, as IC261 inhibited the functional expression of -T692-mENaC. Although CK1, and specifically the CK1 isoform, may selectively influence -T663-hENaC functional and surface expression, the specific context of residue 663 in the COOH terminus is not critical for this effect. These data are thus inconsistent with the hypothesis that differential expression of the A663T-hENaC polymorphism is due to phosphorylation of the -T663 residue by CK1.
Our data also suggest that CK1 exerts its modulatory effect on -T663-hENaC functional and surface expression by influencing the delivery of -T663-hENaC to the oocyte membrane. CK1 inhibition does not affect the rate at which -T663-hENaC is retrieved from the oocyte membrane. These kinetic data contrast with our previous results suggesting that the delivery of -T663-hENaC and -A663-hENaC occurs at similar rates. Furthermore, our previous data suggested that -T663-hENaC's rate of retrieval from the oocyte membrane is slower than that of -A663-hENaC, which results in -T663-hENaC having greater functional and surface expression. Taken together, these results also suggest that CK1 phosphorylation does not underlie the functionality of the -A663T-hENaC polymorphism in the oocyte system.
Although -T663 is predicted to be a substrate for CK1, our data suggest that it is not a substrate for this kinase. We did not observe phosphorylation of -T663, or any other residue within the COOH-terminal 20 amino acids of -hENaC by CK1 in vitro. These data suggest that CK1 exerts its enhancement of -T663-hENaC delivery to the oocyte membrane by an indirect mechanism. This is consistent with the known intracellular localization of this CK1 isoform, where it is found associated with the ER, Golgi, and transport vesicles.
Such indirect regulation of ENaC trafficking by phosphorylation of proteins that interact with ENaC is well described. Perhaps the best example of this is the increase in ENaC functional and surface expression observed after activation of the serum and glucocorticoid regulated kinase, SGK. SGK does not phosphorylate ENaC directly. It phosphorylates the E3-ubiquitin ligase Nedd4-2, which leads to decreased interaction of Nedd4-2 with ENaC and decreased removal of ENaC from the plasma membrane (8, 29). Activation of ENaC by preferential phosphorylation of -T663 nevertheless remains an attractive hypothesis. However, our experiments with CK1 and in previous work with PKC (32) suggest that, if this hypothesis is true, other as yet unidentified kinases are responsible for phosphorylation of -T663.
There is a potential alternate explanation for our not observing direct phosphorylation of the -T663 by CK1 under our experimental conditions. The consensus phosphorylation site for CK1 is S/T-X-X-S/T (with the target of CK1 in bold). Previous data also suggest that phosphorylation of the –3 residue (indicated in italics) or the presence of a group of negatively charged amino acids NH2-terminal of the target significantly enhances the phosphorylation of the target S or T (bold) by CK1, although phosphorylation of targets without such features has been reported (16). In our in vitro experiments, phosphorylation of the italicized –3 residue (S660 in -hENaC) was not present either preceding or after CK1 phosphorylation. Also, the COOH terminus of -hENaC is relatively devoid of acidic residues. Thus these data do not completely exclude the possibility that CK1 could directly modulate -T663-hENaC delivery to the plasma membrane by phosphorylation of the -T663 residue in vivo.
In summary, our data suggest that CK1, and specifically the CK1 isoform, selectively regulates the functional and surface expression of the -T663-hENaC allele of the common polymorphism -A663T in the COOH terminus of -hENaC. This regulation is exerted at the level of hENaC biosynthesis and/or trafficking to the membrane and is distinct from the mechanism by which the -A663T polymorphism alters hENaC functional and surface expression. Also, unlike our previous results regarding the -A663T polymorphism, the context of the COOH-terminal 20 residues of -hENaC is not critical for the effect of CK1. There are at least three potential CK1 targets that could influence ENaC trafficking. 1) CK1 could phosphorylate proteins that modulate ENaC trafficking in a manner that is influenced by the A663T polymorphism. 2) CK1 could directly phosphorylate the COOH terminus of -hENaC at T663. However, phosphorylation of site 3 residues NH2 terminal to T663 would be required for CK1 to phosphorylate T663. 3) CK1 could directly phosphorylate hENaC at a site other than the distal COOH terminus of -hENaC. However, CK1-dependent phosphorylation would be influenced by the A663T polymorphism.
We predict that the alleles of the -A663T polymorphism will have a differential response to signals and stimuli that result in modulation of the activity of the CK1 isoform, or perhaps in a kinase that could phosphorylate S660 and prime -hENaC for CK1 phosphorylation. Such differential regulation may ultimately result in altered hENaC function and consequently altered risk for developing hypertension.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-subunit of the human epithelial sodium channel by protein kinase Cdelta. Am J Physiol Renal Physiol 290: F279–F288, 2006.This article has been cited by other articles:
|
|
 |
|
  V. Bhalla and K. R. Hallows Mechanisms of ENaC Regulation and Clinical Implications J. Am. Soc. Nephrol., October 1, 2008; 19(10): 1845 - 1854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Bachhuber, J. Almaca, F. Aldehni, A. Mehta, M. D. Amaral, R. Schreiber, and K. Kunzelmann Regulation of the Epithelial Na+ Channel by the Protein Kinase CK2 J. Biol. Chem., May 9, 2008; 283(19): 13225 - 13232. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
in B) or presence (open bar in A, 
in B) of 20 µM IC261. After 24–36 h, amiloride-sensitive currents at –100 mV holding potential were determined by TEV (A), and the oocytes were subsequently incubated in MBS solution containing 5 µM Brefeldin A (B). Amiloride-sensitive currents were again determined by TEV after 2, 4, and 6 h of incubation in Brefeldin A. These amiloride-sensitive currents were expressed relative to the amiloride-sensitive current before incubation with Brefeldin A (means ± SE), and apparent first-order rate constants were fit as described in MATERIALS AND METHODS and compared by a 2-tailed t-test. These data were derived from 4 different batches of oocytes.
