Differential modulation of a polymorphism in the COOH terminus of the α-subunit of the human epithelial sodium channel by protein kinase Cδ

Wusheng Yan, Laurence Suaud, Thomas R. Kleyman, Ronald C. Rubenstein

Abstract

The A663T polymorphism of the α-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of αβγ-hENaC in Xenopus laevis oocytes. The context of this residue in the COOH terminus of α-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 α-T663 residue might be phosphorylated by PKCδ. General inhibition of PKC with calphostin C decreased the functional and surface expression of αT663-hENaC and not αA663-hENaC, and was without effect on αA692-mENaC, αT692-mENaC, and a chimeric m(1–678)/h(650–669)αT663, mβγ-ENaC. These data suggest that residues outside of the α-hENaC COOH terminus are important for modulation of αT663-hENaC trafficking by PKC. In contrast, expression of PKCδ decreased the functional and surface expression of αT663-hENaC and the functional expression of m(1–678)/h(650–669)αT663, mβγ-ENaC, and was without effect on αA663-hENaC, αA692-mENaC, or αT692-mENaC. PKCδ did not phosphorylate the COOH terminus of either αT663-hENaC or αA663-hENaC in vitro, suggesting that it acts indirectly to regulate hENaC trafficking. αT663-hENaC was retrieved from the oocyte membrane more slowly than αA663-hENaC, and calphostin C increased the rate of αT663-hENaC removal from the oocyte membrane to a rate similar to that of αA663-hENaC. In contrast, PKCδ did not alter the rate of removal of αT663-hENaC from the oocyte membrane, suggesting that PKCδ altered rates of αT663-hENaC biosynthesis and/or delivery to the plasma membrane. These data are consistent with PKC isoform-specific effects on the intracellular trafficking of αT663- vs. αA663-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 α-, β-, and γ-ENaC, that likely assemble as an α211 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., βT594M) (4), suggesting that ENaC polymorphisms that alter functional channel expression may contribute to the development of hypertension in the general population.

αA663T is a common polymorphism in the COOH terminus of the α-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 αT663βγ-hENaC had significantly higher currents than oocytes expressing αA663βγ 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 α-subunit, a region that is not well conserved between human and mouse α-subunits and that may influence interaction with the cystic fibrosis transmembrane conductance regulator (32). Interestingly, we demonstrated that the αA692T mutation in mouse ENaC (mENaC), corresponding to human αA663T, was not associated differences in functional αβγ-mENaC expression, whereas replacement of the distal COOH terminus of the mouse α-subunit with the distal COOH terminus of the human α-subunit restored the functional differences that were observed with the human αA663T polymorphism.

That αT663 is potentially modifiable by phosphorylation, and from our previous observations that mutation of αT663 to αD663 does not alter the functional expression of hENaC in oocytes (21) suggests the hypothesis that phosphorylation of αT663 may regulate its increased functional and surface expression in X. laevis oocytes. Our data suggest that global inhibition of PKC, and specific expression of PKCδ, can regulate the functional and surface expression of αT663-hENaC, and not αA663-hENaC, and that the context of the distal COOH terminus of the ENaC α-subunit is important for this effect. Our data also suggest that global inhibition of PKC and expression of PKCδ influence the intracellular trafficking of the αT663-hENaC by different mechanisms. Finally, our data suggest that regulation of αT663-hENaC by PKCδ does not result from direct phosphorylation of αT663 by PKCδ and that PKCδ-dependent regulation of αT663-hENaC does not account for the enhanced functional and surface expression of αT663-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 PKCδ in oocytes.

α-, β-, and γ-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 PKCδ and a kinase-dead enhanced green fluorescent protein-murine PKCδ 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 α-hENaC and α-mENaC, wild-type β-hENaC and β-mENaC, wild-type γ-hENaC and γ-mENaC, and PKCδ and kinase-dead PKCδ 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 PKCδ cRNA or 2–20 ng of kinase-dead PKCδ 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 MΩ 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 β-hENaC with a COOH-terminal V5 epitope tag (β-V5) as previously described (11). Briefly, cRNAs for αβ-V5γ-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-α-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 β-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 β-V5-hENaC was assessed by immunoblotting of whole oocyte lysate prepared using the lysis buffer and procedure described above. Whole oocyte expression of PKCδ 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 αT663- and αA663-hENaC were created by blunt-end ligation of AvrII/SphI fragments of the respective α-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-αT663, or GST-αA663 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 PKCδ (Upstate Cell Signaling), and [γ-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 PKCδ 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 γ-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 αT663βγG536C- or αA663βγG536C-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 αT663βγ-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 α-subunit of the hENaC affects functional and surface expression of αβγ-hENaC in X. laevis oocytes. αA663βγ exhibited significantly lower whole cell currents and surface expression compared with αT663βγ. This polymorphism is located at the COOH terminus of α-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 α-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 α-mENaC (679–699) with those of α-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 αT663 may regulate the functional and surface expression of αT663-hENaC.

Fig. 1.

Amino acid sequences of the COOH termini of human (h), mouse (Mur), and rat α-subunit of the epithelial sodium channel (ENaC; α-ENaC). Amino acid residue numbers at the beginning and end of each sequence are indicated. Gray shading indicates identity. Residue 663 of α-hENaC and its homologous residue (692) in α-mENaC and rat α-ENaC are highlighted in black.

We therefore queried a phosphoprotein prediction database to ask whether αT663 was a predicted substrate for a known kinase. A query in scansite.mit.edu v1.5 suggested that the αT663 residue might be a substrate for phosphorylation by PKCδ. Interestingly, αT692-mENaC was not predicted to be a substrate of PKCδ 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 αT663- and αA663-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 β-V5-hENaC (Fig. 2B) or whole oocyte expression as assessed by immunoblot of β-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.

Fig. 2.

PKC activation reduces hENaC functional expression without altering surface expression in oocytes. A: αT663- or αA663-hENaC was expressed in oocytes, and 2-electrode voltage clamp (TEV) was performed before and after treatment with 200 nM PMA for 20 min. Amiloride-sensitive whole cell currents were determined at −100-mV holding potential (adjusted for resting membrane potential). Values are means ± SE, with the P value determined by a paired t-test as described in materials and methods. B: hαT663βγ- or αA663βγ-ENaC, where the β-subunit had a COOH-terminal V5 epitope tag (β-V5), was expressed in oocytes. Cell surface biotinylation was performed as described in materials and methods either without or with exposure to 200 nM PMA for 20 min, and β-V5-hENaC was recovered by streptavidin-agarose precipitation and quantitated by immunoblotting with an anti-V5 antibody. A representative immunoblot (n = 3 independent experiments) is shown. C: representative immunoblots of whole oocyte lysates of αT663- and αA663-injected oocytes demonstrating similar expression of β-V5-hENaC after treatment with 200 nM PMA for 20 min are also shown and are similarly representative of n = 3 independent experiments.

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 αT663- and αA663-hENaC in oocytes. Oocytes were injected with αT663- or αA663-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 αT663-hENaC but does not alter the functional expression of αA663-hENaC in oocytes. This pattern of αT663- and αA663-hENaC functional expression after exposure to calphostin C corresponded to the surface expression of these hENaCs. Whole oocyte expression of β-V5-hENaC was unaltered by calphostin C for αT663- and αA663-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 αT663βγ-hENaC in oocytes.

Fig. 3.

PKC inhibition selectively reduces αT663-hENaC functional and surface expression in oocytes. A: αT663βγ- or αA663βγ-hENaC was expressed in oocytes, and TEV was performed after incubation in the absence (gray bars) or presence (open bars) of 200 nM calphostin C for 24–48 h. Amiloride-sensitive whole cell currents were determined at −100-mV holding potential (adjusted for resting membrane potential) and are expressed relative to the mean whole oocyte current for αT663βγ-hENaC-expressing oocytes. Values are means ± SE with the P value determined by 1-way ANOVA as described in materials and methods. B: hαT663βγ- or αA663βγ-ENaC, where the β-subunit had a COOH-terminal V5 epitope tag (β-V5), was expressed in oocytes in the absence or presence of 200 nM calphostin C for 24–48 h. Cell surface biotinylation was performed as described under in materials and methods, and β-V5-hENaC was recovered by streptavidin-agarose precipitation and quantitated by immunoblotting with an anti-V5 antibody. A representative immunoblot (n = 5 independent experiments) is shown. A representative immunoblot of whole oocyte lysates of αT663- and αA663-injected oocytes demonstrating similar expression of β-V5-hENaC is also shown and is similarly representative of n = 5 independent experiments. C: densitometric analysis of the surface biotinylation experiments was performed as described in materials and methods and are expressed as means ± SE of n = 5 independent experiments, normalized to values obtained with oocytes expressing αT663β-V5γ-hENaC. Open bars indicate oocytes incubated in the presence of 200 nM calphostin C for 24–48 h after injection, whereas oocytes represented by gray bars were incubated under control conditions. P values were determined by ANOVA compared with αT663β-V5γ-hENaC.

If a member of PKC family is “the” kinase that causes differential functional and surface expression of the αA663T-hENaC polymorphism, then we predict that calphostin C would not influence the functional expression of αA692T-mENaC but would alter the functional expression of the chimeric m(1–678)/h(650–669)αA663Tmβγ-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 αT692- or αA692-mENaC, which is consistent with the notion that the context of αT692 (homologous to αT663 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 α-hENaC, as the m(1–678)/h(650–669)αT663mβγ-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 α, β1, β2, γ, δ, ζ, and ε) (14, 31) may selectively regulate the αA663T-hENaC functional polymorphism, these data suggest that residues outside of the COOH-terminal 20 amino acids of α-hENaC may influence this regulation.

Fig. 4.

Influence of PKC inhibition on mENaC and chimeric ENaC in oocytes. αT692- or αA692mβγ-ENaC (A) or m(1–678)/h(650–669)αT663mβγ (m-hαT663)- or m(1–678)/h(650–669)αA663mβγ (m-hαA663)-ENaC (B) chimera was expressed in oocytes, and TEV was performed after incubation in the absence (gray bars) or presence (open bars) of 200 nM calphostin C for 24–48 h. Amiloride-sensitive whole cell currents were determined at −100-mV holding potential (adjusted for resting membrane potential) and are expressed relative to the mean whole oocyte current for αT692-mβγ-ENaC (A)- or m(1–678)/h(650–669)αT663mβγ-ENaC (B) chimera-expressing oocytes. Values are means ± SE, with the P value determined by 2-tailed t-test as described in materials and methods. ns, Not significant.

Influence of PKC δ on hENaC functional and surface expression.

We then tested the hypothesis that the αA663T-hENaC functional polymorphism might be selectively regulated by PKCδ. PKCδ was readily expressed in oocytes when 5 ng of PKCδ cRNA were coinjected with αT663- or αA663-hENaC (Fig. 5A). Although endogenous PKCδ 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 PKCδ cRNA was coinjected with αT663- or αA663-hENaC, expression of PKCδ decreased the functional expression of αT663-, but not αA663-hENaC (Fig. 5B). This pattern of functional expression of αT663- and αA663-hENaC in response to PKCδ expression was again consistent with the surface expression of β-V5 when PKCδ was coinjected with either αT663β-V5γ-hENaC or αA663β-V5γ-hENaC (Fig. 5C; densitometric quantitation in Fig. 5D), suggesting that PKCδ was primarily influencing the trafficking of αT663-hENaC without affecting the trafficking of αA663-hENaC. Coinjection of PKCδ with αβ-V5γ-hENaC did not alter whole oocyte expression of β-V5-hENaC (Fig. 5C), suggesting that PKCδ does not alter the steady-state whole oocyte expression of β-V5-hENaC and that competition for oocyte translational machinery does not confound these experiments.

Fig. 5.

PKCδ expression selectively reduces αT663-hENaC functional and surface expression in oocytes. A: αT663- or αA663-hENaC cRNA was injected into oocytes either alone or together with 5 ng cRNA for PKCδ. Whole oocyte lysates were prepared, and PKCδ was detected by immunoblotting. B: αT663- or αA663-hENaC cRNA was injected into oocytes either alone (gray bars) or coinjected with 5 ng cRNA for PKCδ (open bars), and TEV was performed after 24–48 h. Amiloride-sensitive whole cell currents were determined at −100-mV holding potential (adjusted for resting membrane potential) and are expressed relative to the mean whole oocyte current for αT663βγ-hENaC-expressing oocytes. Values are means ± SE, with the P value determined by 1-way ANOVA as described in materials and methods. C: αT663βγ- or αA663βγ-hENaC, where the β-subunit had a COOH-terminal V5 epitope tag (β-V5), was expressed in oocytes either without or with coinjection of 5 ng PKC δ cRNA. Cell surface biotinylation was performed as described in materials and methods, and β-V5-hENaC was recovered by streptavidin-agarose precipitation and quantitated by immunoblotting with an anti-V5 antibody. A representative immunoblot (n = 5 independent experiments) is shown. A representative immunoblot of whole oocyte lysates of αT663- and αA663-injected oocytes demonstrating similar expression of β-V5-hENaC is also shown and is similarly representative of n = 5 independent experiments. D: densitometric analysis of the surface biotinylation experiments was performed as described in materials and methods and are expressed as means ± SE, n = 5 independent experiments, normalized to values obtained with oocytes expressing αT663β-V5γ-hENaC. Open bars indicate oocytes coinjected with ENaC and 5 ng PKCδ cRNA, whereas oocytes represented by gray bars were injected with ENaC alone. P values were determined by ANOVA compared with αT663β-V5γ-hENaC.

We again tested the specificity of PKCδ's regulation of αT663 for the context of the COOH-terminal 20 amino acids of α-hENaC by assessing the influence of PKCδ on the functional expression of αA692T-mENaC and the m(1–678)/h(650–669)αA663mβγ-ENaC chimera. Here, expression of PKCδ had no effect on the functional expression of either αT692-mENaC or αA692-mENaC (Fig. 6A), whereas the m(1–678)/h(650–669)αT663mβγ-ENaC chimera, but not the m(1–678)/h(650–669)αA663mβγ-ENaC chimera, had decreased functional expression when PKCδ was coinjected. Thus unlike our data on calphostin C, our observations here are consistent with PKCδ exerting its selective influence on αT663-hENaC functional and surface expression in the specific context of the 20 COOH-terminal residues of α-hENaC.

Fig. 6.

Influence of PKCδ expression on mENaC and chimeric ENaC in oocytes. αT692- or αA692βγ-mENaC (A) or m(1–678)/h(650–669)αT663βγ (m-hαT663)- or m(1–678)/h(650–669)αA663mβγ (m-hαA663)-ENaC chimera (B) cRNA was injected either alone (gray bars) or was coinjected with PKCδ cRNA (5 ng). Amiloride-sensitive whole cell currents were determined 24–48 h after injection at −100-mV holding potential (adjusted for resting membrane potential) and are expressed relative to the mean whole oocyte current for αT692βγ-mENaC (A) or m(1–678)/h(650–669)αT663mβγ-ENaC (B) chimera-expressing oocytes. Values are means ± SE, with the P value determined by 2-tailed t-test as described in materials and methods.

We also assessed whether the kinase activity of PKCδ was required for this effect by coinjecting a kinase-dead PKCδ. Figure 7 demonstrates essentially unaltered functional expression of αT663-hENaC with coinjection of increasing amounts of cRNA for a kinase-dead PKCδ. These data also serve as an additional control to suggest that competition for translational machinery does not confound these experiments.

Fig. 7.

Kinase-dead PKCδ expression does not influence αT663-hENaC functional expression in oocytes. Oocytes were injected with cRNA for α-T663βγ-hENaC (2 ng/subunit) and the indicated amount of cRNA for a kinase-dead (K472N) PKCδ. After 24–48 h, whole oocyte amiloride-sensitive currents at −100-mV holding potential (adjusted for baseline transmembrane potential) were measured by TEV. Values are means ± SE expressed relative to the mean whole oocyte current for oocytes injected with αT663βγ-hENaC alone with statistical significance assessed by ANOVA as described in materials and methods.

PKCδ does not directly phosphorylate the COOH terminus of α-hENaC.

We next aimed to assess whether PKCδ would selectively phosphorylate the αT663 residue of hENaC. To facilitate these experiments, we constructed GST fusion proteins containing the COOH-terminal 20 residues of αT663- and αA663-hENaC; as demonstrated above, these 20 residues are sufficient to confer selective regulation of ENaC by PKCδ. As shown in Fig. 8, neither GST, GST-αT663, nor GST-αA663 was phosphorylated in vitro by active PKCδ. 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 PKCδ expression selectively regulates the intracellular trafficking of α-T663-hENaC by an indirect mechanism rather than by direct phosphorylation of the αT663 residue.

Fig. 8.

PKCδ does not phosphorylate αT663 in vitro. GST and COOH-terminal fusions of glutathione S-transferase (GST) containing the COOH-terminal 20 amino acids of α-hENaC, GST-αT663, and GST-αA663 were expressed in Escherichia coli, immobilized on glutathione-Sepharose 4B, and subject to in vitro phosphorylation with PKCδ and [γ-32P]ATP as described in materials and methods. GST, GST-αT663, and GST-αA663 were subsequently resolved by SDS-PAGE. A: Coomassie blue-stained SDS-PAGE demonstrating expression of GST, GST-αT663, and GST-αA663. B: fluorogram (3-day exposure) of A demonstrating lack of 32P associated with GST, GST-αT663, and GST-αA663. The mobility of GST is indicated by the black arrowhead in the left margin, and the mobility of GST-αT663 and GST-αA663 are indicated by the gray arrowhead in the right margin. In A and B, the mobility of the molecular weight standards are indicated in the left margin. C: representative positive control phosphorylation of neurogranin(28-43) by active PKCδ was performed using the SignaTECT kit according to the manufacturer's protocol as described in materials and methods. These data are presented as counts/minute determined by liquid scintillation counting and are the average of duplicate determinations. Data in AC are each representative of n = 3 independent experiments.

Influence of the αA663T polymorphism on hENaC trafficking in oocytes.

To better understand the mechanism by which the αA663T polymorphism alters the functional and surface expression of hENaC in oocytes, we sought to determine whether the increased functional and surface expression of αT663-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 γ-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 αA663T polymorphism does not influence the rate of hENaC delivery to the plasma membrane in oocytes.

Fig. 9.

αA663T polymorphism influences hENaC intracellular trafficking. A: to assess the rate of hENaC delivery to the oocyte membrane, oocytes were injected with either αA663βγG536C- or αT663βγG536C-hENaC. After 24 h, benzamil (100 μM)-sensitive currents were determined by TEV before, during, and after covalent blockade of these channels with 1 mM MTSET as described in materials and methods. Values are means ± SE presented as the benzamil-sensitive current relative to that of the initial benzamil-sensitive current to account for batch-to-batch and oocyte-to-oocyte variability. These data are derived from 5 different batches of oocytes. B: to assess the rate of removal of hENaC from the oocyte membrane, oocytes were injected with αA663βγ (triangles)- or αT663βγ-hENaC (squares). After 24–36 h, amiloride-sensitive currents were determined by TEV, and the oocytes were subsequently incubated in 5 μM brefeldin A. 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.

In contrast, the αA663T 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 αA663-hENaC functional expression (k = −0.28 ± 0.02 h−1, n = 20) was significantly greater than that of αT663-hENaC (k = −0.21 ± 0.02 h−1, n = 20, P = 0.027). These data are consistent with the αA663T 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 PKCδ expression on the removal of αT663-hENaC from the oocyte membrane.

As shown above, both global inhibition of PKC with calphostin C (Fig. 3) and expression of PKCδ (Fig. 5) reduced functional and surface expression of αT663-hENaC. As this result seemed paradoxical, we sought further mechanistic insight by assessing the influence of calphostin C and PKCδ expression on the rate of retrieval of αT663-hENaC from the oocyte plasma membrane. Global inhibition of PKC with calphostin C increased the rate constant for αT663-hENaC removal from the oocyte membrane (Fig. 10A). Interestingly, the rate constant for αT663-hENaC removal from the oocyte membrane in the presence of calphostin was similar to that for the removal of αA663-hENaC (Fig. 9B). In contrast, coexpression of PKCδ and αT663-hENaC did not alter the rate constant for removal of αT663-hENaC from the oocyte plasma membrane (Fig. 10B), suggesting that PKCδ affects rates of biosynthesis and/or delivery of αT663-hENaC to the plasma membrane.

Fig. 10.

Differential effects of PKC inhibition and PKCδ expression on the removal of αT663 from the oocyte membrane. The apparent first-order rate constant for removal of αT663-hENaC from the oocyte membrane was determined as described in materials and methods and the legend for Fig. 9B for oocytes injected with αT663-hENaC and incubated in the absence (squares) or presence (circles) of 200 nM calphostin C for 24–36 h prior to assay (A) or oocytes injected with αT663-hENaC either alone (squares) or coinjected with 5 ng cRNA for PKCδ (circles; B). Values are means ± SE presented as amiloride-sensitive currents relative to the initial amiloride-sensitive current for each individual oocyte and result from 5 and 3 batches of oocytes for A and B, respectively.

DISCUSSION

We have previously demonstrated that a COOH-terminal functional polymorphism of hENaC, αA663T was associated with decreased functional and surface expression in X. laevis oocytes and that the context of the COOH-terminal 20 amino acids of α-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 α-hENaC. 1) The intervention should congruently alter functional and surface expression of either αT663- or αA663-hENaC, but not both. 2) The intervention should not influence the functional expression of αT692- or αA692-mENaC, as this α-A663T-homologous change in mENaC does not alter mENaC functional expression (21). 3) Replacement of the COOH-terminal 21 amino acids of α-mENaC with the COOH-terminal 20 amino acids of α-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 α-hENaC. 4) If phosphorylation of αT663-hENaC is responsible for the increase in channel activity, activation of the appropriate kinase should selectivity increase the activity of αT663-hENaC.

That αT663 is potentially modified by phosphorylation, and our previous data that mutation of αT663 to αD663 does not alter the functional expression of hENaC in oocytes (21), led us to test the hypothesis that phosphorylation of αT663 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 αT663 by PKCδ, so we specifically tested the hypotheses that the action of PKC and, specifically PKCδ might regulate the increased functional and surface expression of αT663- vs. αA663-hENaC via the context of the COOH-terminal 20 amino acids of α-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 β- and γ-subunit expression (5, 29). In contrast, PKC activation reduces levels of α-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 αT663- and αA663-hENaC, thus failing criterion 1. In contrast global, tonic inhibition of PKC with calphostin C decreased the functional and surface expression of αT663-hENaC without influencing αA663-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 αT692- or αA692-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)α-T663mβγ-ENaC chimera. These data suggest that, whereas a PKC may influence αT663-hENaC functional and surface expression, this effect is not mediated solely through the COOH-terminal 20 amino acids of αT663-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 α COOH terminus. The present studies do not directly address which calphostin C-inhibited PKC isoform(s) endogenous within oocytes differentially regulates hENaC (αT663) and mENaC (αT692).

In contrast, our data regarding the influence of PKCδ 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 α-ENaC. Expression of PKCδ selectively decreased αT663-hENaC functional and surface expression but did not alter αA663-hENaC functional or surface expression. Expression of PKCδ also did not influence the functional expression of either αT692- or αA692-mENaC, and introduction of the COOH-terminal 20 amino acids of α-hENaC into mENaC [m(1–678)/h(650–669)α-T663mβγ-ENaC] resurrected the influence of PKCδ expression. These data are therefore consistent with our hypothesis that PKCδ selectively influences the trafficking of αT663-hENaC, but not αA663-hENaC, in the context of the COOH-terminal 20 residues of αT663-hENaC.

While consistent with our hypothesis that PKCδ would selectively regulate the trafficking of αT663-hENaC, but not αA663-hENaC, the decrease in functional and surface expression of αT663-hENaC caused by expression of PKCδ failed criterion 4. Our prediction, based on our published data that αT663D-hENaC had essentially the same functional expression in oocytes as αT663-hENaC (21), was that selective phosphorylation of αT663 would increase αT663-hENaC trafficking and functional expression. The selective regulation of αT663-hENaC vs. αA663-hENaC by PKCδ suggests that stimuli and signaling pathways that alter PKCδ activity in epithelial cells may selectively affect the activity of αT663-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 PKCδ, αT663 is in fact not a substrate for this kinase, and that PKCδ influences αT663-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 αT663 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 αT663-hENaC, relative to that of αA663-hENaC, is a result of a reduced rate of removal of αT663-hENaC from the plasma membrane. Inhibition of endogenous PKC activity by calphostin C led to an increase in the rate of removal of αT663-hENaC from the plasma membrane, suggesting than one (or more) active PKC isoforms are modulating rates of αT663-hENaC endocytosis. Expression of PKCδ did not affect rates of αT663-hENaC endocytosis, suggesting that 1) PKCδ is not one of the PKC isoforms that modulates rates of αT663-hENaC endocytosis and 2) PKCδ-dependent inhibition of αT663-hENaC reflects a reduced rate of biosynthesis and/or delivery of αT663-hENaC to the plasma membrane.

In summary, our data suggest that PKCδ selectively regulates the functional and surface expression of the αT663-hENaC allele of the common polymorphism αA663T in the COOH terminus of α-hENaC and that the context of the COOH-terminal 20 residues of α-hENaC are important for this effect. Furthermore, our results with calphostin C suggest that other PKC isoforms selectively modulate αT663-hENaC surface expression. We predict that the alleles of the αA663T 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

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REFERENCES

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