Differential modulation of a polymorphism in the COOH terminus of the -subunit of the human epithelial sodium channel by protein k
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《美国生理学杂志》
1Division of Pulmonary Medicine, The Children's Hospital of Philadelphia
2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia
3Departments of Medicine and of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania
ABSTRACT
The A663T polymorphism of the -subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of -hENaC in Xenopus laevis oocytes. The context of this residue in the COOH terminus of -hENaC is important for this effect, as a homologous change in murine ENaC (mENaC), A692T, does not alter functional and surface expression of mENaC. Query of a phosphoprotein database suggested that the -T663 residue might be phosphorylated by PKC. General inhibition of PKC with calphostin C decreased the functional and surface expression of T663-hENaC and not A663-hENaC, and was without effect on A692-mENaC, T692-mENaC, and a chimeric m(1–678)/h(650–669)T663, m-ENaC. These data suggest that residues outside of the -hENaC COOH terminus are important for modulation of T663-hENaC trafficking by PKC. In contrast, expression of PKC decreased the functional and surface expression of T663-hENaC and the functional expression of m(1–678)/h(650–669)T663, m-ENaC, and was without effect on A663-hENaC, A692-mENaC, or T692-mENaC. PKC did not phosphorylate the COOH terminus of either T663-hENaC or A663-hENaC in vitro, suggesting that it acts indirectly to regulate hENaC trafficking. T663-hENaC was retrieved from the oocyte membrane more slowly than A663-hENaC, and calphostin C increased the rate of T663-hENaC removal from the oocyte membrane to a rate similar to that of A663-hENaC. In contrast, PKC did not alter the rate of removal of T663-hENaC from the oocyte membrane, suggesting that PKC altered rates of T663-hENaC biosynthesis and/or delivery to the plasma membrane. These data are consistent with PKC isoform-specific effects on the intracellular trafficking of T663- vs. A663-hENaC.
trafficking; oocyte; phosphorylation
EPITHELIAL SODIUM CHANNELS (ENaCs) are expressed in principal cells in the late distal convoluted tubule, connecting tubule, and collecting tubule, where they serve as a final site for reabsorption of Na+ from the glomerular ultrafiltrate. Volume-regulatory hormones, such as aldosterone, have a key role in modifying rates of renal tubular Na+ reabsorption through regulation of functional ENaC expression at the apical plasma membrane (15). ENaCs are also found in airway epithelia, where their hyperfunction is hypothesized to contribute to the pathophysiology of impaired mucociliary clearance and chronic respiratory infections in cystic fibrosis.
ENaCs are composed of three structurally related subunits, termed -, -, and -ENaC, that likely assemble as an 2,1,1 tetramer (10, 17), although alternate subunit stoichiometries have been proposed (25). The three subunits have limited (30–40%) sequence identity but share a common topology of two membrane-spanning domains and intracellular NH2 and COOH termini (6, 20, 26).
Changes in ENaC functional expression are associated with alterations in blood pressure (12, 13). ENaC loss-of-function mutations lead to type I pseudohypoaldosteronism, a disorder characterized by volume depletion, hypotension, and hyperkalemia (8, 22), as well as profuse respiratory secretions and increased mucociliary clearance (16). In contrast, ENaC gain-of-function mutations cause Liddle's syndrome, a disorder characterized by volume expansion, hypokalemia, and hypertension (24) but, interestingly, little pulmonary phenotype (3). Some common human ENaC (hENaC) polymorphisms may segregate with blood pressure (i.e., T594M) (4), suggesting that ENaC polymorphisms that alter functional channel expression may contribute to the development of hypertension in the general population.
A663T is a common polymorphism in the COOH terminus of the -subunit of hENaC, and there are conflicting data reported as to whether this polymorphism segregates with blood pressure (1, 30). We have previously shown that Xenopus laevis oocytes expressing wild-type T663-hENaC had significantly higher currents than oocytes expressing A663 and that these the higher currents were associated with higher levels of cell surface expression of channels, suggesting that this polymorphism altered channel trafficking (21). 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 that may influence interaction with the cystic fibrosis transmembrane conductance regulator (32). Interestingly, we demonstrated that the A692T mutation in mouse ENaC (mENaC), corresponding to human A663T, was not associated 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.
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. Our data suggest that global inhibition of PKC, and specific expression of PKC, can regulate the functional and surface expression of T663-hENaC, and not A663-hENaC, and that the context of the distal COOH terminus of the ENaC -subunit is important for this effect. Our data also suggest that global inhibition of PKC and expression of PKC influence the intracellular trafficking of the T663-hENaC by different mechanisms. Finally, our data suggest that regulation of T663-hENaC by PKC does not result from direct phosphorylation of T663 by PKC and that PKC-dependent regulation of T663-hENaC does not account for the enhanced functional and surface expression of T663-hENaC.
MATERIALS AND METHODS
Materials. Calphostin C was purchased from Calbiochem (La Jolla, CA). All other reagents were purchased from Fisher Chemical.
Expression of ENaC and PKC in oocytes. -, -, and -hENaC cDNAs were from M. J. Welsh (University of Iowa). 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, 32). cDNAs for murine PKC and a kinase-dead enhanced green fluorescent protein-murine PKC fusion protein (K472N; active site lysine replaced by asparagine) were a gift of Dr. C. Stubbs (Jefferson Medical College).
cRNAs for wild-type and mutant -hENaC and -mENaC, wild-type -hENaC and -mENaC, wild-type -hENaC and -mENaC, and PKC and kinase-dead PKC 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, 5 ng of PKC cRNA or 2–20 ng of kinase-dead PKC cRNA were coinjected with the hENaC cRNAs. After injection, oocytes were incubated at 18°C in modified Barth's saline [MBS; (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 15 HEPES, 0.3 Ca (NO3)2, 0.41 CaCl2, 0.82 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, a protein kinase inhibitor was added to the MBS immediately after injection. The animal protocol was approved by the Children's Hospital of Philadelphia's and the University of Pittsburgh's Institutional Animal Care and Use Committees.
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 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 a resistance 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 (in mM) 100 Na gluconate, 2 KCl, 1.8 CaCl2, 3 BaCl2, 10 tetraethylammonium Cl, and 10 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 have previously described (21, 32). To facilitate detection of biotinylated hENaC subunits, these experiments used a -hENaC with a COOH-terminal V5 epitope tag (-V5) 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/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 was assessed by immunoblotting of whole oocyte lysate prepared using the lysis buffer and procedure described above. Whole oocyte expression of PKC was similarly detected by immunoblot of whole oocyte lysate using an antibody purchased from BD Biosciences.
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 immunoblotting, suggesting that our oocytes were not leaky after mechanical stripping of the vitelline membrane.
In vitro phosphorylation. COOH-terminal glutathione S-transferase (GST) fusion proteins containing the COOH-terminal 20 amino acids of T663- and A663-hENaC were created by blunt-end ligation of AvrII/SphI fragments of the respective -hENaC plasmids into the EcoRI site of pGEX4T2 (Amersham). Orientation and in-frame translation were confirmed by automated DNA sequencing in The Children's Hospital of Philadelphia Nucleic Acid and Protein Core.
GST, GST-T663, or GST-A663 was expressed in Escherichia coli BL21, immobilized on glutathione-Sepharose 4B beads (Amersham), and subject to in vitro phosphorylation using the SignaTECT PKC assay system buffers, active PKC (Upstate Cell Signaling), and [-32P]ATP (10 μCi, 3,000 Ci/mmol, DuPont New England Nuclear). Bound GST or GST fusion proteins were eluted by boiling 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 biotinylated neurogranin(28-43) provided in the SignaTECT PKC assay kit for PKC was assayed in parallel according to the manufacturer's protocol. In each experiment, phosphorylation of the neurogranin substrate was increased at least 10-fold over control (by liquid scintillation counting), suggesting that the lack of phosphorylation of the GST or GST fusion proteins was not due to a problem in the assay.
Assessment of hENaC delivery to and removal from the oocyte plasma membrane. To assess the rate of delivery of hENaC to the oocyte membrane, we made use of the observations that ENaC mutants where a cysteine was introduced into the "amiloride binding site" 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) was used for these studies as channels containing amiloride binding site mutations are relatively amiloride insensitive (7, 27). We therefore constructed the G536C mutation in -hENaC using the Quik-Change kit (Promega) and confirmed its sequence by automated sequencing the Children's Hospital of Philadelphia Nucleic Acid and Protein Core. Oocytes were then injected with T663G536C- or A663G536C-hENaC as described above. Twenty-four to thirty-six hours after injection, whole oocyte currents were determined by TEV before application of MTSET, after 2 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 insensitive 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 means 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 (except those in Fig. 2) were normalized by the mean amiloride-sensitive current for the control condition (usually T663-hENaC) within a batch of oocytes before the combining of 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. 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 Gaussian distribution (34). A P value 0.05 was considered significant. We also independently analyzed the statistical significance of these data without normalization and transformation using the Wilcoxon rank sum/Mann-Whitney U-test for nonparametric results, and obtained similar P values to the method outlined above (data not shown). Other data that were normally distributed (including those in Fig. 2) 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
We have previously demonstrated that the A663T polymorphism of the -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 compared with T663. 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 homologous change in mENaC, A692T does not alter functional and surface expression of mENaC, but 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 recently published by our group (21), we hypothesized that phosphorylation of T663 may regulate the functional and surface expression of T663-hENaC.
We therefore queried a phosphoprotein prediction database to ask whether T663 was a predicted substrate for a known kinase. A query in scansite.mit.edu v1.5 suggested that the T663 residue might be a substrate for phosphorylation by PKC. Interestingly, T692-mENaC was not predicted to be a substrate of PKC in a similar query, which is consistent with our previous observations that the context of this polymorphism is important for its functional effects (21).
Influence of PKC activation on hENaC functional and surface expression. We first assessed the influence of acute activation of PKC with PMA on the functional expression of hENaC in oocytes. Consistent with the data of others (2), acute, nonspecific activation of PKC with PMA caused a decrease in amiloride-sensitive current, or functional expression of both T663- and A663-hENaC in oocytes (Fig. 2A). This decrease in functional expression was not associated with a change in hENaC surface expression as assessed by surface biotinylation of -V5-hENaC (Fig. 2B) or whole oocyte expression as assessed by immunoblot of -V5-hENaC (Fig. 2C). These data are thus consistent with acute, nonspecific activation of PKC altering ENaC open probability (Po) or unitary conductance in oocytes.
Influence of PKC inhibition on hENaC functional and surface expression. We next assessed the influence of tonic inhibition of PKC on the functional and surface expression of T663- and A663-hENaC in oocytes. Oocytes were injected with T663- or A663-hENaC and then incubated either with, or without 200 nM calphostin C, a non-isotype-selective PKC inhibitor that binds to the PKC diacyl glycerol binding site, for 24–48 h. As shown in Fig. 3A, calphostin C decreases the ENaC functional expression in oocytes injected with T663-hENaC but does not alter the functional expression of A663-hENaC in oocytes. This pattern of T663- and A663-hENaC functional expression after exposure to calphostin C corresponded to the surface expression of these hENaCs. Whole oocyte expression of -V5-hENaC was unaltered by calphostin C for T663- and A663-hENaC (Fig. 3B; densitometry of the surface biotinylation experiments is shown in Fig. 3C). These data are thus consistent with PKC selectively regulating the trafficking of T663-hENaC in oocytes.
If a member of PKC family is "the" kinase that causes differential functional and surface expression of the A663T-hENaC polymorphism, then we predict that calphostin C would not influence the functional expression of A692T-mENaC but would alter the functional expression of the chimeric m(1–678)/h(650–669)A663Tm-ENaC (21). Experiments testing these predictions are shown in Fig. 4. Figure 4A demonstrates that calphostin C does not influence the functional expression of either T692- or A692-mENaC, which is consistent with the notion that the context of T692 (homologous to T663 in humans) is critical for inhibition of PKC by calphostin C to have functional effects. However, Fig. 4B suggests that the effect of calphostin C cannot be resurrected in mENaC by the COOH-terminal 20 residues of -hENaC, as the m(1–678)/h(650–669)T663m-ENaC chimera has unaltered functional expression in the presence of calphostin C. Thus, whereas the family of PKCs (oocytes have been reported to express PKCs , 1, 2, , , , and ) (14, 31) may selectively regulate the A663T-hENaC functional polymorphism, these data suggest that residues outside of the COOH-terminal 20 amino acids of -hENaC may influence this regulation.
Influence of PKC on hENaC functional and surface expression. We then tested the hypothesis that the A663T-hENaC functional polymorphism might be selectively regulated by PKC. PKC was readily expressed in oocytes when 5 ng of PKC cRNA were coinjected with T663- or A663-hENaC (Fig. 5A). Although endogenous PKC expression has been reported in oocytes (as detected by immunoblot) (14, 31), we did not detect such endogenous expression under our experimental conditions (Fig. 5A). When PKC cRNA was coinjected with T663- or A663-hENaC, expression of PKC decreased the functional expression of T663-, but not A663-hENaC (Fig. 5B). This pattern of functional expression of T663- and A663-hENaC in response to PKC expression was again consistent with the surface expression of -V5 when PKC was coinjected with either T663-V5-hENaC or A663-V5-hENaC (Fig. 5C; densitometric quantitation in Fig. 5D), suggesting that PKC was primarily influencing the trafficking of T663-hENaC without affecting the trafficking of A663-hENaC. Coinjection of PKC with -V5-hENaC did not alter whole oocyte expression of -V5-hENaC (Fig. 5C), suggesting that PKC does not alter the steady-state whole oocyte expression of -V5-hENaC and that competition for oocyte translational machinery does not confound these experiments.
We again tested the specificity of PKC's regulation of T663 for the context of the COOH-terminal 20 amino acids of -hENaC by assessing the influence of PKC on the functional expression of A692T-mENaC and the m(1–678)/h(650–669)A663m-ENaC chimera. Here, expression of PKC had no effect on the functional expression of either T692-mENaC or A692-mENaC (Fig. 6A), whereas the m(1–678)/h(650–669)T663m-ENaC chimera, but not the m(1–678)/h(650–669)A663m-ENaC chimera, had decreased functional expression when PKC was coinjected. Thus unlike our data on calphostin C, our observations here are consistent with PKC exerting its selective influence on T663-hENaC functional and surface expression in the specific context of the 20 COOH-terminal residues of -hENaC.
We also assessed whether the kinase activity of PKC was required for this effect by coinjecting a kinase-dead PKC. Figure 7 demonstrates essentially unaltered functional expression of T663-hENaC with coinjection of increasing amounts of cRNA for a kinase-dead PKC. These data also serve as an additional control to suggest that competition for translational machinery does not confound these experiments.
PKC does not directly phosphorylate the COOH terminus of -hENaC. We next aimed to assess whether PKC would selectively phosphorylate the T663 residue of hENaC. To facilitate these experiments, we constructed GST fusion proteins containing the COOH-terminal 20 residues of T663- and A663-hENaC; as demonstrated above, these 20 residues are sufficient to confer selective regulation of ENaC by PKC. As shown in Fig. 8, neither GST, GST-T663, nor GST-A663 was phosphorylated in vitro by active PKC. Parallel positive control experiments performed under the same reaction conditions demonstrated robust phosphorylation of a model substrate, neurogranin(28–43), suggesting that the lack of phosphorylation of the GST fusion proteins was not a result of a problem with the assay system. These data suggest that PKC expression selectively regulates the intracellular trafficking of -T663-hENaC by an indirect mechanism rather than by direct phosphorylation of the T663 residue.
Influence of the A663T polymorphism on hENaC trafficking in oocytes. To better understand the mechanism by which the A663T polymorphism alters the functional and surface expression of hENaC in oocytes, we sought to determine whether the increased functional and surface expression of T663-hENaC was due to an increased rate of delivery of hENaC to the membrane or a reduced rate of its removal from the membrane. To assess the rate of delivery, we introduced a cysteine residue into the amiloride binding site of the -hENaC subunit (G536C); such mutants allow irreversible block of the hENaC channel after treatment with MTSET (27). The rate of recovery of benzamil-sensitive current (with benzamil being used because the mutant channels are relatively insensitive to amiloride) (7) is then a direct measure of the rate of hENaC delivery to the oocyte membrane. These data are shown in Fig. 9A and suggest that the A663T polymorphism does not influence the rate of hENaC delivery to the plasma membrane in oocytes.
In contrast, the A663T polymorphism decreased the rate at which hENaC was removed from the plasma membrane in oocytes (Fig. 9B). In the presence of brefeldin A to block the delivery of new hENaC channels to the oocyte membrane, the apparent first-order rate constant for loss of A663-hENaC functional expression (k = –0.28 ± 0.02 h–1, n = 20) was significantly greater than that of T663-hENaC (k = –0.21 ± 0.02 h–1, n = 20, P = 0.027). These data are consistent with the A663T polymorphism influencing the functional and surface expression of hENaC in oocytes by altering the rate of channel removal from the oocyte membrane.
Differential effects of PKC inhibition and PKC expression on the removal of T663-hENaC from the oocyte membrane. As shown above, both global inhibition of PKC with calphostin C (Fig. 3) and expression of PKC (Fig. 5) reduced functional and surface expression of T663-hENaC. As this result seemed paradoxical, we sought further mechanistic insight by assessing the influence of calphostin C and PKC expression on the rate of retrieval of T663-hENaC from the oocyte plasma membrane. Global inhibition of PKC with calphostin C increased the rate constant for T663-hENaC removal from the oocyte membrane (Fig. 10A). Interestingly, the rate constant for T663-hENaC removal from the oocyte membrane in the presence of calphostin was similar to that for the removal of A663-hENaC (Fig. 9B). In contrast, coexpression of PKC and T663-hENaC did not alter the rate constant for removal of T663-hENaC from the oocyte plasma membrane (Fig. 10B), suggesting that PKC affects rates of biosynthesis and/or delivery of T663-hENaC to the plasma membrane.
DISCUSSION
We have previously demonstrated that a COOH-terminal functional polymorphism of hENaC, A663T was associated with decreased functional and surface expression in X. laevis oocytes and that the context of the COOH-terminal 20 amino acids of -hENaC was critical for this effect (21). To begin to assess the molecular basis underlying the functional effect of this polymorphism, and based on our previous observations (21), we 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. 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 this -A663T-homologous change in mENaC does not alter mENaC functional expression (21). 3) Replacement of the COOH-terminal 21 amino acids of -mENaC with the COOH-terminal 20 amino acids of -hENaC in a chimeric ENaC should resurrect the effect of the intervention on the ENaC chimera, and this effect should be the same as that observed for hENaC. Satisfying this third criterion would thereby demonstrate 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.
That T663 is potentially modified by phosphorylation, and our previous data that mutation of T663 to D663 does not alter the functional expression of hENaC in oocytes (21), led us to test the hypothesis that phosphorylation of T663 in hENaC may regulate its increased functional and surface expression in X. laevis oocytes. Query of a protein phosphorylation prediction database suggested potential phosphorylation of T663 by PKC, so we specifically tested the hypotheses that the action of 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.
Activation of PKC by phorbol esters inhibits ENaC activity in renal epithelia (5, 18, 19, 33). In A6 cells, phorbol ester-dependent activation of PKC results in a rapid inhibition of ENaC, and these inhibitory effects are maintained over 24–28 h. While the rapid inhibition of ENaC activity reflect a reduction in channel Po, the long-term inhibitory effects of phorbol esters are due to a MAPK/ERK1/2 dependent reduction in the levels of - and -subunit expression (5, 29). In contrast, PKC activation reduces levels of -subunit expression in parotid cells via a MAPK/ERK-dependent pathway (35).
We observed that global, acute activation of PKC by a phorbol ester did not yield differential effects on T663- and A663-hENaC, thus failing criterion 1. In contrast global, tonic inhibition of PKC with calphostin C decreased the functional and surface expression of T663-hENaC without influencing A663-hENaC, satisfying criterion 1. Inhibition of PKC with chronic calphostin C exposure also satisfied criterion 2, as there was essentially no effect of calphostin C on the functional expression of either T692- or A692-mENaC. However, the effect of calphostin C did not satisfy criterion 3, as its effect on the mENaC background is not observed in the m(1–678)/h(650–669)-T663m-ENaC chimera. These data suggest that, whereas a PKC may influence T663-hENaC functional and surface expression, this effect is not mediated solely through the COOH-terminal 20 amino acids of T663-hENaC.
One can speculate about a few potential mechanisms by which this might occur. For example, calphostin C might inhibit more than one isoform of PKC that differentially regulates hENaC and mENaC at sites distinct from the COOH terminus. The present studies do not directly address which calphostin C-inhibited PKC isoform(s) endogenous within oocytes differentially regulates hENaC (T663) and mENaC (T692).
In contrast, our data regarding the influence of PKC expression do satisfy our first three criteria for selective modulation of functional and surface expression of this polymorphic hENaC in the context of the COOH-terminal 20 amino acids of -ENaC. Expression of PKC selectively decreased T663-hENaC functional and surface expression but did not alter A663-hENaC functional or surface expression. Expression of PKC also did not influence the functional expression of either T692- or A692-mENaC, and introduction of the COOH-terminal 20 amino acids of -hENaC into mENaC [m(1–678)/h(650–669)-T663m-ENaC] resurrected the influence of PKC expression. These data are therefore consistent with our hypothesis that PKC selectively influences the trafficking of T663-hENaC, but not A663-hENaC, in the context of the COOH-terminal 20 residues of T663-hENaC.
While consistent with our hypothesis that PKC would selectively regulate the trafficking of T663-hENaC, but not A663-hENaC, the decrease in functional and surface expression of T663-hENaC caused by expression of PKC failed criterion 4. Our prediction, based on our published data that T663D-hENaC had essentially the same functional expression in oocytes as T663-hENaC (21), was that selective phosphorylation of T663 would increase T663-hENaC trafficking and functional expression. The selective regulation of T663-hENaC vs. A663-hENaC by PKC suggests that stimuli and signaling pathways that alter PKC activity in epithelial cells may selectively affect the activity of T663-hENaC and thereby potentially modulate a susceptibility to hypertension conferred by this polymorphism.
Our data also suggest that, even though it is predicted to be a substrate for PKC, T663 is in fact not a substrate for this kinase, and that PKC influences T663-hENaC trafficking through an indirect mechanism. Such regulation of ENaC trafficking by phosphorylation of proteins that interact with ENaC rather than ENaC itself is well described, with the best example being the increase in ENaC functional and surface expression on activation of the serum and glucocorticoid regulated kinase (SGK). SGK, rather than phosphorylating ENaC directly, 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 (9, 28). While other kinases might activate ENaC by preferentially phosphorylating T663 in a species-specific manner, these remain to be identified.
Our experiments examining rates of delivery and removal of hENaC from the plasma membrane suggest that the increase in surface expression of T663-hENaC, relative to that of A663-hENaC, is a result of a reduced rate of removal of T663-hENaC from the plasma membrane. Inhibition of endogenous PKC activity by calphostin C led to an increase in the rate of removal of T663-hENaC from the plasma membrane, suggesting than one (or more) active PKC isoforms are modulating rates of T663-hENaC endocytosis. Expression of PKC did not affect rates of T663-hENaC endocytosis, suggesting that 1) PKC is not one of the PKC isoforms that modulates rates of T663-hENaC endocytosis and 2) PKC-dependent inhibition of T663-hENaC reflects a reduced rate of biosynthesis and/or delivery of T663-hENaC to the plasma membrane.
In summary, our data suggest that PKC selectively regulates the functional and surface expression of the T663-hENaC allele of the common polymorphism A663T in the COOH terminus of -hENaC and that the context of the COOH-terminal 20 residues of -hENaC are important for this effect. Furthermore, our results with calphostin C suggest that other PKC isoforms selectively modulate T663-hENaC surface expression. We predict that the alleles of the A663T polymorphism will have a differential response to signals and stimuli that result in activation of specific PKC isoforms. Such differential regulation may ultimately result in altered hENaC function and, consequently, altered risk for developing hypertension.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54354 and DK-58046. R. C. Rubenstein is an Established Investigator of the American Heart Association.
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.
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2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia
3Departments of Medicine and of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania
ABSTRACT
The A663T polymorphism of the -subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of -hENaC in Xenopus laevis oocytes. The context of this residue in the COOH terminus of -hENaC is important for this effect, as a homologous change in murine ENaC (mENaC), A692T, does not alter functional and surface expression of mENaC. Query of a phosphoprotein database suggested that the -T663 residue might be phosphorylated by PKC. General inhibition of PKC with calphostin C decreased the functional and surface expression of T663-hENaC and not A663-hENaC, and was without effect on A692-mENaC, T692-mENaC, and a chimeric m(1–678)/h(650–669)T663, m-ENaC. These data suggest that residues outside of the -hENaC COOH terminus are important for modulation of T663-hENaC trafficking by PKC. In contrast, expression of PKC decreased the functional and surface expression of T663-hENaC and the functional expression of m(1–678)/h(650–669)T663, m-ENaC, and was without effect on A663-hENaC, A692-mENaC, or T692-mENaC. PKC did not phosphorylate the COOH terminus of either T663-hENaC or A663-hENaC in vitro, suggesting that it acts indirectly to regulate hENaC trafficking. T663-hENaC was retrieved from the oocyte membrane more slowly than A663-hENaC, and calphostin C increased the rate of T663-hENaC removal from the oocyte membrane to a rate similar to that of A663-hENaC. In contrast, PKC did not alter the rate of removal of T663-hENaC from the oocyte membrane, suggesting that PKC altered rates of T663-hENaC biosynthesis and/or delivery to the plasma membrane. These data are consistent with PKC isoform-specific effects on the intracellular trafficking of T663- vs. A663-hENaC.
trafficking; oocyte; phosphorylation
EPITHELIAL SODIUM CHANNELS (ENaCs) are expressed in principal cells in the late distal convoluted tubule, connecting tubule, and collecting tubule, where they serve as a final site for reabsorption of Na+ from the glomerular ultrafiltrate. Volume-regulatory hormones, such as aldosterone, have a key role in modifying rates of renal tubular Na+ reabsorption through regulation of functional ENaC expression at the apical plasma membrane (15). ENaCs are also found in airway epithelia, where their hyperfunction is hypothesized to contribute to the pathophysiology of impaired mucociliary clearance and chronic respiratory infections in cystic fibrosis.
ENaCs are composed of three structurally related subunits, termed -, -, and -ENaC, that likely assemble as an 2,1,1 tetramer (10, 17), although alternate subunit stoichiometries have been proposed (25). The three subunits have limited (30–40%) sequence identity but share a common topology of two membrane-spanning domains and intracellular NH2 and COOH termini (6, 20, 26).
Changes in ENaC functional expression are associated with alterations in blood pressure (12, 13). ENaC loss-of-function mutations lead to type I pseudohypoaldosteronism, a disorder characterized by volume depletion, hypotension, and hyperkalemia (8, 22), as well as profuse respiratory secretions and increased mucociliary clearance (16). In contrast, ENaC gain-of-function mutations cause Liddle's syndrome, a disorder characterized by volume expansion, hypokalemia, and hypertension (24) but, interestingly, little pulmonary phenotype (3). Some common human ENaC (hENaC) polymorphisms may segregate with blood pressure (i.e., T594M) (4), suggesting that ENaC polymorphisms that alter functional channel expression may contribute to the development of hypertension in the general population.
A663T is a common polymorphism in the COOH terminus of the -subunit of hENaC, and there are conflicting data reported as to whether this polymorphism segregates with blood pressure (1, 30). We have previously shown that Xenopus laevis oocytes expressing wild-type T663-hENaC had significantly higher currents than oocytes expressing A663 and that these the higher currents were associated with higher levels of cell surface expression of channels, suggesting that this polymorphism altered channel trafficking (21). 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 that may influence interaction with the cystic fibrosis transmembrane conductance regulator (32). Interestingly, we demonstrated that the A692T mutation in mouse ENaC (mENaC), corresponding to human A663T, was not associated 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.
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. Our data suggest that global inhibition of PKC, and specific expression of PKC, can regulate the functional and surface expression of T663-hENaC, and not A663-hENaC, and that the context of the distal COOH terminus of the ENaC -subunit is important for this effect. Our data also suggest that global inhibition of PKC and expression of PKC influence the intracellular trafficking of the T663-hENaC by different mechanisms. Finally, our data suggest that regulation of T663-hENaC by PKC does not result from direct phosphorylation of T663 by PKC and that PKC-dependent regulation of T663-hENaC does not account for the enhanced functional and surface expression of T663-hENaC.
MATERIALS AND METHODS
Materials. Calphostin C was purchased from Calbiochem (La Jolla, CA). All other reagents were purchased from Fisher Chemical.
Expression of ENaC and PKC in oocytes. -, -, and -hENaC cDNAs were from M. J. Welsh (University of Iowa). 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, 32). cDNAs for murine PKC and a kinase-dead enhanced green fluorescent protein-murine PKC fusion protein (K472N; active site lysine replaced by asparagine) were a gift of Dr. C. Stubbs (Jefferson Medical College).
cRNAs for wild-type and mutant -hENaC and -mENaC, wild-type -hENaC and -mENaC, wild-type -hENaC and -mENaC, and PKC and kinase-dead PKC 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, 5 ng of PKC cRNA or 2–20 ng of kinase-dead PKC cRNA were coinjected with the hENaC cRNAs. After injection, oocytes were incubated at 18°C in modified Barth's saline [MBS; (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 15 HEPES, 0.3 Ca (NO3)2, 0.41 CaCl2, 0.82 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, a protein kinase inhibitor was added to the MBS immediately after injection. The animal protocol was approved by the Children's Hospital of Philadelphia's and the University of Pittsburgh's Institutional Animal Care and Use Committees.
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 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 a resistance 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 (in mM) 100 Na gluconate, 2 KCl, 1.8 CaCl2, 3 BaCl2, 10 tetraethylammonium Cl, and 10 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 have previously described (21, 32). To facilitate detection of biotinylated hENaC subunits, these experiments used a -hENaC with a COOH-terminal V5 epitope tag (-V5) 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/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 was assessed by immunoblotting of whole oocyte lysate prepared using the lysis buffer and procedure described above. Whole oocyte expression of PKC was similarly detected by immunoblot of whole oocyte lysate using an antibody purchased from BD Biosciences.
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 immunoblotting, suggesting that our oocytes were not leaky after mechanical stripping of the vitelline membrane.
In vitro phosphorylation. COOH-terminal glutathione S-transferase (GST) fusion proteins containing the COOH-terminal 20 amino acids of T663- and A663-hENaC were created by blunt-end ligation of AvrII/SphI fragments of the respective -hENaC plasmids into the EcoRI site of pGEX4T2 (Amersham). Orientation and in-frame translation were confirmed by automated DNA sequencing in The Children's Hospital of Philadelphia Nucleic Acid and Protein Core.
GST, GST-T663, or GST-A663 was expressed in Escherichia coli BL21, immobilized on glutathione-Sepharose 4B beads (Amersham), and subject to in vitro phosphorylation using the SignaTECT PKC assay system buffers, active PKC (Upstate Cell Signaling), and [-32P]ATP (10 μCi, 3,000 Ci/mmol, DuPont New England Nuclear). Bound GST or GST fusion proteins were eluted by boiling 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 biotinylated neurogranin(28-43) provided in the SignaTECT PKC assay kit for PKC was assayed in parallel according to the manufacturer's protocol. In each experiment, phosphorylation of the neurogranin substrate was increased at least 10-fold over control (by liquid scintillation counting), suggesting that the lack of phosphorylation of the GST or GST fusion proteins was not due to a problem in the assay.
Assessment of hENaC delivery to and removal from the oocyte plasma membrane. To assess the rate of delivery of hENaC to the oocyte membrane, we made use of the observations that ENaC mutants where a cysteine was introduced into the "amiloride binding site" 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) was used for these studies as channels containing amiloride binding site mutations are relatively amiloride insensitive (7, 27). We therefore constructed the G536C mutation in -hENaC using the Quik-Change kit (Promega) and confirmed its sequence by automated sequencing the Children's Hospital of Philadelphia Nucleic Acid and Protein Core. Oocytes were then injected with T663G536C- or A663G536C-hENaC as described above. Twenty-four to thirty-six hours after injection, whole oocyte currents were determined by TEV before application of MTSET, after 2 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 insensitive 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 means 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 (except those in Fig. 2) were normalized by the mean amiloride-sensitive current for the control condition (usually T663-hENaC) within a batch of oocytes before the combining of 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. 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 Gaussian distribution (34). A P value 0.05 was considered significant. We also independently analyzed the statistical significance of these data without normalization and transformation using the Wilcoxon rank sum/Mann-Whitney U-test for nonparametric results, and obtained similar P values to the method outlined above (data not shown). Other data that were normally distributed (including those in Fig. 2) 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
We have previously demonstrated that the A663T polymorphism of the -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 compared with T663. 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 homologous change in mENaC, A692T does not alter functional and surface expression of mENaC, but 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 recently published by our group (21), we hypothesized that phosphorylation of T663 may regulate the functional and surface expression of T663-hENaC.
We therefore queried a phosphoprotein prediction database to ask whether T663 was a predicted substrate for a known kinase. A query in scansite.mit.edu v1.5 suggested that the T663 residue might be a substrate for phosphorylation by PKC. Interestingly, T692-mENaC was not predicted to be a substrate of PKC in a similar query, which is consistent with our previous observations that the context of this polymorphism is important for its functional effects (21).
Influence of PKC activation on hENaC functional and surface expression. We first assessed the influence of acute activation of PKC with PMA on the functional expression of hENaC in oocytes. Consistent with the data of others (2), acute, nonspecific activation of PKC with PMA caused a decrease in amiloride-sensitive current, or functional expression of both T663- and A663-hENaC in oocytes (Fig. 2A). This decrease in functional expression was not associated with a change in hENaC surface expression as assessed by surface biotinylation of -V5-hENaC (Fig. 2B) or whole oocyte expression as assessed by immunoblot of -V5-hENaC (Fig. 2C). These data are thus consistent with acute, nonspecific activation of PKC altering ENaC open probability (Po) or unitary conductance in oocytes.
Influence of PKC inhibition on hENaC functional and surface expression. We next assessed the influence of tonic inhibition of PKC on the functional and surface expression of T663- and A663-hENaC in oocytes. Oocytes were injected with T663- or A663-hENaC and then incubated either with, or without 200 nM calphostin C, a non-isotype-selective PKC inhibitor that binds to the PKC diacyl glycerol binding site, for 24–48 h. As shown in Fig. 3A, calphostin C decreases the ENaC functional expression in oocytes injected with T663-hENaC but does not alter the functional expression of A663-hENaC in oocytes. This pattern of T663- and A663-hENaC functional expression after exposure to calphostin C corresponded to the surface expression of these hENaCs. Whole oocyte expression of -V5-hENaC was unaltered by calphostin C for T663- and A663-hENaC (Fig. 3B; densitometry of the surface biotinylation experiments is shown in Fig. 3C). These data are thus consistent with PKC selectively regulating the trafficking of T663-hENaC in oocytes.
If a member of PKC family is "the" kinase that causes differential functional and surface expression of the A663T-hENaC polymorphism, then we predict that calphostin C would not influence the functional expression of A692T-mENaC but would alter the functional expression of the chimeric m(1–678)/h(650–669)A663Tm-ENaC (21). Experiments testing these predictions are shown in Fig. 4. Figure 4A demonstrates that calphostin C does not influence the functional expression of either T692- or A692-mENaC, which is consistent with the notion that the context of T692 (homologous to T663 in humans) is critical for inhibition of PKC by calphostin C to have functional effects. However, Fig. 4B suggests that the effect of calphostin C cannot be resurrected in mENaC by the COOH-terminal 20 residues of -hENaC, as the m(1–678)/h(650–669)T663m-ENaC chimera has unaltered functional expression in the presence of calphostin C. Thus, whereas the family of PKCs (oocytes have been reported to express PKCs , 1, 2, , , , and ) (14, 31) may selectively regulate the A663T-hENaC functional polymorphism, these data suggest that residues outside of the COOH-terminal 20 amino acids of -hENaC may influence this regulation.
Influence of PKC on hENaC functional and surface expression. We then tested the hypothesis that the A663T-hENaC functional polymorphism might be selectively regulated by PKC. PKC was readily expressed in oocytes when 5 ng of PKC cRNA were coinjected with T663- or A663-hENaC (Fig. 5A). Although endogenous PKC expression has been reported in oocytes (as detected by immunoblot) (14, 31), we did not detect such endogenous expression under our experimental conditions (Fig. 5A). When PKC cRNA was coinjected with T663- or A663-hENaC, expression of PKC decreased the functional expression of T663-, but not A663-hENaC (Fig. 5B). This pattern of functional expression of T663- and A663-hENaC in response to PKC expression was again consistent with the surface expression of -V5 when PKC was coinjected with either T663-V5-hENaC or A663-V5-hENaC (Fig. 5C; densitometric quantitation in Fig. 5D), suggesting that PKC was primarily influencing the trafficking of T663-hENaC without affecting the trafficking of A663-hENaC. Coinjection of PKC with -V5-hENaC did not alter whole oocyte expression of -V5-hENaC (Fig. 5C), suggesting that PKC does not alter the steady-state whole oocyte expression of -V5-hENaC and that competition for oocyte translational machinery does not confound these experiments.
We again tested the specificity of PKC's regulation of T663 for the context of the COOH-terminal 20 amino acids of -hENaC by assessing the influence of PKC on the functional expression of A692T-mENaC and the m(1–678)/h(650–669)A663m-ENaC chimera. Here, expression of PKC had no effect on the functional expression of either T692-mENaC or A692-mENaC (Fig. 6A), whereas the m(1–678)/h(650–669)T663m-ENaC chimera, but not the m(1–678)/h(650–669)A663m-ENaC chimera, had decreased functional expression when PKC was coinjected. Thus unlike our data on calphostin C, our observations here are consistent with PKC exerting its selective influence on T663-hENaC functional and surface expression in the specific context of the 20 COOH-terminal residues of -hENaC.
We also assessed whether the kinase activity of PKC was required for this effect by coinjecting a kinase-dead PKC. Figure 7 demonstrates essentially unaltered functional expression of T663-hENaC with coinjection of increasing amounts of cRNA for a kinase-dead PKC. These data also serve as an additional control to suggest that competition for translational machinery does not confound these experiments.
PKC does not directly phosphorylate the COOH terminus of -hENaC. We next aimed to assess whether PKC would selectively phosphorylate the T663 residue of hENaC. To facilitate these experiments, we constructed GST fusion proteins containing the COOH-terminal 20 residues of T663- and A663-hENaC; as demonstrated above, these 20 residues are sufficient to confer selective regulation of ENaC by PKC. As shown in Fig. 8, neither GST, GST-T663, nor GST-A663 was phosphorylated in vitro by active PKC. Parallel positive control experiments performed under the same reaction conditions demonstrated robust phosphorylation of a model substrate, neurogranin(28–43), suggesting that the lack of phosphorylation of the GST fusion proteins was not a result of a problem with the assay system. These data suggest that PKC expression selectively regulates the intracellular trafficking of -T663-hENaC by an indirect mechanism rather than by direct phosphorylation of the T663 residue.
Influence of the A663T polymorphism on hENaC trafficking in oocytes. To better understand the mechanism by which the A663T polymorphism alters the functional and surface expression of hENaC in oocytes, we sought to determine whether the increased functional and surface expression of T663-hENaC was due to an increased rate of delivery of hENaC to the membrane or a reduced rate of its removal from the membrane. To assess the rate of delivery, we introduced a cysteine residue into the amiloride binding site of the -hENaC subunit (G536C); such mutants allow irreversible block of the hENaC channel after treatment with MTSET (27). The rate of recovery of benzamil-sensitive current (with benzamil being used because the mutant channels are relatively insensitive to amiloride) (7) is then a direct measure of the rate of hENaC delivery to the oocyte membrane. These data are shown in Fig. 9A and suggest that the A663T polymorphism does not influence the rate of hENaC delivery to the plasma membrane in oocytes.
In contrast, the A663T polymorphism decreased the rate at which hENaC was removed from the plasma membrane in oocytes (Fig. 9B). In the presence of brefeldin A to block the delivery of new hENaC channels to the oocyte membrane, the apparent first-order rate constant for loss of A663-hENaC functional expression (k = –0.28 ± 0.02 h–1, n = 20) was significantly greater than that of T663-hENaC (k = –0.21 ± 0.02 h–1, n = 20, P = 0.027). These data are consistent with the A663T polymorphism influencing the functional and surface expression of hENaC in oocytes by altering the rate of channel removal from the oocyte membrane.
Differential effects of PKC inhibition and PKC expression on the removal of T663-hENaC from the oocyte membrane. As shown above, both global inhibition of PKC with calphostin C (Fig. 3) and expression of PKC (Fig. 5) reduced functional and surface expression of T663-hENaC. As this result seemed paradoxical, we sought further mechanistic insight by assessing the influence of calphostin C and PKC expression on the rate of retrieval of T663-hENaC from the oocyte plasma membrane. Global inhibition of PKC with calphostin C increased the rate constant for T663-hENaC removal from the oocyte membrane (Fig. 10A). Interestingly, the rate constant for T663-hENaC removal from the oocyte membrane in the presence of calphostin was similar to that for the removal of A663-hENaC (Fig. 9B). In contrast, coexpression of PKC and T663-hENaC did not alter the rate constant for removal of T663-hENaC from the oocyte plasma membrane (Fig. 10B), suggesting that PKC affects rates of biosynthesis and/or delivery of T663-hENaC to the plasma membrane.
DISCUSSION
We have previously demonstrated that a COOH-terminal functional polymorphism of hENaC, A663T was associated with decreased functional and surface expression in X. laevis oocytes and that the context of the COOH-terminal 20 amino acids of -hENaC was critical for this effect (21). To begin to assess the molecular basis underlying the functional effect of this polymorphism, and based on our previous observations (21), we 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. 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 this -A663T-homologous change in mENaC does not alter mENaC functional expression (21). 3) Replacement of the COOH-terminal 21 amino acids of -mENaC with the COOH-terminal 20 amino acids of -hENaC in a chimeric ENaC should resurrect the effect of the intervention on the ENaC chimera, and this effect should be the same as that observed for hENaC. Satisfying this third criterion would thereby demonstrate 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.
That T663 is potentially modified by phosphorylation, and our previous data that mutation of T663 to D663 does not alter the functional expression of hENaC in oocytes (21), led us to test the hypothesis that phosphorylation of T663 in hENaC may regulate its increased functional and surface expression in X. laevis oocytes. Query of a protein phosphorylation prediction database suggested potential phosphorylation of T663 by PKC, so we specifically tested the hypotheses that the action of 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.
Activation of PKC by phorbol esters inhibits ENaC activity in renal epithelia (5, 18, 19, 33). In A6 cells, phorbol ester-dependent activation of PKC results in a rapid inhibition of ENaC, and these inhibitory effects are maintained over 24–28 h. While the rapid inhibition of ENaC activity reflect a reduction in channel Po, the long-term inhibitory effects of phorbol esters are due to a MAPK/ERK1/2 dependent reduction in the levels of - and -subunit expression (5, 29). In contrast, PKC activation reduces levels of -subunit expression in parotid cells via a MAPK/ERK-dependent pathway (35).
We observed that global, acute activation of PKC by a phorbol ester did not yield differential effects on T663- and A663-hENaC, thus failing criterion 1. In contrast global, tonic inhibition of PKC with calphostin C decreased the functional and surface expression of T663-hENaC without influencing A663-hENaC, satisfying criterion 1. Inhibition of PKC with chronic calphostin C exposure also satisfied criterion 2, as there was essentially no effect of calphostin C on the functional expression of either T692- or A692-mENaC. However, the effect of calphostin C did not satisfy criterion 3, as its effect on the mENaC background is not observed in the m(1–678)/h(650–669)-T663m-ENaC chimera. These data suggest that, whereas a PKC may influence T663-hENaC functional and surface expression, this effect is not mediated solely through the COOH-terminal 20 amino acids of T663-hENaC.
One can speculate about a few potential mechanisms by which this might occur. For example, calphostin C might inhibit more than one isoform of PKC that differentially regulates hENaC and mENaC at sites distinct from the COOH terminus. The present studies do not directly address which calphostin C-inhibited PKC isoform(s) endogenous within oocytes differentially regulates hENaC (T663) and mENaC (T692).
In contrast, our data regarding the influence of PKC expression do satisfy our first three criteria for selective modulation of functional and surface expression of this polymorphic hENaC in the context of the COOH-terminal 20 amino acids of -ENaC. Expression of PKC selectively decreased T663-hENaC functional and surface expression but did not alter A663-hENaC functional or surface expression. Expression of PKC also did not influence the functional expression of either T692- or A692-mENaC, and introduction of the COOH-terminal 20 amino acids of -hENaC into mENaC [m(1–678)/h(650–669)-T663m-ENaC] resurrected the influence of PKC expression. These data are therefore consistent with our hypothesis that PKC selectively influences the trafficking of T663-hENaC, but not A663-hENaC, in the context of the COOH-terminal 20 residues of T663-hENaC.
While consistent with our hypothesis that PKC would selectively regulate the trafficking of T663-hENaC, but not A663-hENaC, the decrease in functional and surface expression of T663-hENaC caused by expression of PKC failed criterion 4. Our prediction, based on our published data that T663D-hENaC had essentially the same functional expression in oocytes as T663-hENaC (21), was that selective phosphorylation of T663 would increase T663-hENaC trafficking and functional expression. The selective regulation of T663-hENaC vs. A663-hENaC by PKC suggests that stimuli and signaling pathways that alter PKC activity in epithelial cells may selectively affect the activity of T663-hENaC and thereby potentially modulate a susceptibility to hypertension conferred by this polymorphism.
Our data also suggest that, even though it is predicted to be a substrate for PKC, T663 is in fact not a substrate for this kinase, and that PKC influences T663-hENaC trafficking through an indirect mechanism. Such regulation of ENaC trafficking by phosphorylation of proteins that interact with ENaC rather than ENaC itself is well described, with the best example being the increase in ENaC functional and surface expression on activation of the serum and glucocorticoid regulated kinase (SGK). SGK, rather than phosphorylating ENaC directly, 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 (9, 28). While other kinases might activate ENaC by preferentially phosphorylating T663 in a species-specific manner, these remain to be identified.
Our experiments examining rates of delivery and removal of hENaC from the plasma membrane suggest that the increase in surface expression of T663-hENaC, relative to that of A663-hENaC, is a result of a reduced rate of removal of T663-hENaC from the plasma membrane. Inhibition of endogenous PKC activity by calphostin C led to an increase in the rate of removal of T663-hENaC from the plasma membrane, suggesting than one (or more) active PKC isoforms are modulating rates of T663-hENaC endocytosis. Expression of PKC did not affect rates of T663-hENaC endocytosis, suggesting that 1) PKC is not one of the PKC isoforms that modulates rates of T663-hENaC endocytosis and 2) PKC-dependent inhibition of T663-hENaC reflects a reduced rate of biosynthesis and/or delivery of T663-hENaC to the plasma membrane.
In summary, our data suggest that PKC selectively regulates the functional and surface expression of the T663-hENaC allele of the common polymorphism A663T in the COOH terminus of -hENaC and that the context of the COOH-terminal 20 residues of -hENaC are important for this effect. Furthermore, our results with calphostin C suggest that other PKC isoforms selectively modulate T663-hENaC surface expression. We predict that the alleles of the A663T polymorphism will have a differential response to signals and stimuli that result in activation of specific PKC isoforms. Such differential regulation may ultimately result in altered hENaC function and, consequently, altered risk for developing hypertension.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54354 and DK-58046. R. C. Rubenstein is an Established Investigator of the American Heart Association.
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.
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