ENaC activity and expression is decreased in the lungs of protein kinase C-α knockout mice

Abstract

We used a PKC-α knockout model to investigate the regulation of alveolar epithelial Na(+) channels (ENaC) by PKC. Primary alveolar type II (ATII) cells were subjected to cell-attached patch clamp. In the absence of PKC-α, the open probability (Po) of ENaC was decreased by half compared with wild-type mice. The channel density (N) was also reduced in the knockout mice. Using in vivo biotinylation, membrane localization of all three ENaC subunits (α, β, and γ) was decreased in the PKC-α knockout lung, compared with the wild-type. Confocal microscopy of lung slices showed elevated levels of reactive oxygen species (ROS) in the lungs of the PKC-α knockout mice vs. the wild-type. High levels of ROS in the knockout lung can be explained by a decrease in both cytosolic and mitochondrial superoxide dismutase activity. Elevated levels of ROS in the knockout lung activates PKC-δ and leads to reduced dephosphorylation of ERK1/2 by MAP kinase phosphatase, which in turn causes increased internalization of ENaC via ubiquitination by the ubiquitin-ligase Nedd4-2. In addition, in the knockout lung, PKC-δ activates ERK, causing a decrease in ENaC density at the apical alveolar membrane. PKC-δ also phosphorylates MARCKS, leading to a decrease in ENaC Po. The effects of ROS and PKC-δ were confirmed with patch-clamp experiments on isolated ATII cells in which the ROS scavenger, Tempol, or a PKC-δ-specific inhibitor added to patches reversed the observed decrease in ENaC apical channel density and Po. These results explain the decrease in ENaC activity in PKC-α knockout lung.

Keywords: alveoli; epithelial Na+ channels; knockout mice; lung fluid balance; protein kinase C-α; single channels.

Fig. 1.

Membrane epithelial Na⁺ channels (ENaC) are decreased in the protein kinase C (PKC)-α knockout (KO) lung. A: Western blots of total lung homogenates from wild-type (WT) and PKC-α knockout mice, demonstrating that there is no PKC-α present in the knockout lung. The expected molecular weight is 80–82 kDa. 25 μg of lysate were loaded per well. B: Western blots of total lung homogenate from wild-type and PKC-α knockout mice illustrating that there is no significant difference in total amount of ENaC present in the 2 groups. C: biotinylated ENaC subunits. Whole lungs were homogenized, and protein was extracted. The membrane fraction was equally loaded onto streptavidin beads and incubated overnight. After being washed, protein was eluted with sample buffer, resolved on gels, and detected with ENaC-specific antibodies. The amount of each of the biotinylated subunits was less in the PKC-α knockout lungs than in the wild-type. D: ratio of the band densities in lungs from knockout animals vs. wild-type. Each ratio is <1, indicating that ENaC subunits are reduced in knockout animals (α, *P = 0.031; β, *P = 0.046; γ, *P = 0.039 by 1 sample t-test; average of 3 experiments for each type of animal).

Fig. 2.

Sodium transport is decreased in the PKC-α knockout lung. We quantified physiological alveolar fluid clearance (AFC) using the wet/dry weight assay and the Evan's blue assay. We found that the knockout value was increased over the wild-type for the wet:dry weight assay (A). The value for knockout was significantly reduced compared with the wild-type for the Evan's blue assay as well (B). *Both of these values indicate that overall sodium transport is reduced in the PKC-α knockout lung compared with wild-type.

Fig. 3.

ENaC channel activity is decreased in the PKC-α knockout lung. ENaC channel activity was recorded from cell-attached patches on alveolar type II cells from wild-type and PKC-α knockout mice. All recordings were made at −20 mV (the difference in potential between the inside of the cell and the patch pipette). Asterisks indicate significant differences (P < 0.05) as determined using a t-test. A: the upper 2 traces are long representative records from knockout or wild-type cells (pipette potential is 40 mV). The activity of the knockout cell is substantially lower than that of the wild-type. The expanded regions are magnified 10-fold to emphasize the disparity in activity between the 2 groups. B: current-voltage relationship for the channel in A. The inward rectification and positive reversal potential are characteristic of ENaC. A summary of the single-channel data is shown in C–F. The graph in C shows that ENaC activity, measured as the product of the number of channels (N) times the open probability (P_o), decreases more than 3-fold in the PKC-α knockout mice compared with wild-type (P < 0.002 by rank sum test). When the individual components of activity are examined, both P_o (D) and N (E) decrease significantly in the knockout animals (for P_o, P = 0.025; for N, P = 0.028, Mann-Whitney test). It is important when considering channel density that greater than 5-fold more empty patches were recorded from PKC-α knockout cells vs. wild-type (F, P < 0.001 by z-test). Numbers with the bars represent the number of individual patches made to determine the values for different parameters.

Fig. 4.

Reactive oxygen species (ROS) levels are elevated in the PKC-α knockout lung. 125-μm lung slices were prepared from wild-type and PKC-α knockout mice. Subsequently, the slices were incubated in dihydroethidium (DHE), a fluorescent superoxide reporter, and Erythrina crista-galli lectin (ECL), an alveolar type I cell marker. The 2 panels are divided into 4 images: white-light image overlaid with DHE and ECL (top, left), DHE and ECL (top, right), DHE alone (bottom, left), and white-light image overlaid with DHE (bottom, left). White scale bar is 20 μm and is the same for all images. A: wild-type mice have lower levels of superoxide production than the knockout animals, as shown in B. DHE fluorescence was analyzed using confocal microscopy at excitation/emission 520/610 nm, and data were quantified using Image J. The intensity of the DHE fluorescence over the entire field was 2-fold greater in the knockout animals over the wild-type (P < 0.001 by t-test, n = 3 separate animals for each type). C and D: superoxide dismutase (SOD) activity is decreased in the PKC-α knockout lung. We measured both cytosolic and mitochondrial SOD activity using Superoxide Dismutase Assay Kit (Cayman Chemical). Samples were analyzed using a plate reader at excitation/emission 440/460 nm. Asterisks indicate significant differences as determined by t-test (P < 0.05). C: shows a significant decrease in cytosolic SOD activity in the knockout mice compared with wild-type (P = 0.034, 6 samples per mouse from 3 mice of each type). D: there is 2-fold decrease in mitochondrial SOD activity in the knockout lung vs. wild-type (P < 0.001; 6 samples per mouse from 3 mice of each type).

Fig. 5.

Active ERK1/2 is increased in the PKC-α knockout lung. Using Western blotting, we measured total ERK1/2 and phosphoERK1/2 in whole lung homogenates from PKC-α knockout and wild-type mice (A). We used Image J to quantify the blots using an area that included both ERK1 and ERK2 bands. The program calculated the average density of the bands above the background for individual bands. B: the mean densitometry values from 3 separate experiments illustrating that total ERK is the same (P = 0.159), but phosphoERK is significantly increased in the knockout lung over the wild-type (*P = 0.020; n = 1 blot from 3 mice of each type).

Fig. 6.

Cytosolic MARCKS is increased in the PKC-α knockout lung. Using Western blotting, we measured total MARCKS and phosphoMARCKS in whole-lung homogenates from PKC-α knockout and wild-type mice (A). Unphosphorylated MARCKS protein is at the membrane, facilitating interactions that increase ENaC P_o. When MARCKS is phosphorylated, it moves into the cytosol, causing a decrease in ENaC P_o. We used Image J to quantify the blots. The program calculated the cumulative sum of the pixel values above the background for individual bands. Asterisks indicate significant differences as determined using a t-test (P < 0.05). B: mean densitometry values from 2 separate experiments, which show that total MARCKS is the same (P = n.s.), but phosphoMARCKS is significantly increased in the knockout lung over the wild-type (*P = 0.002; n = 1 blot from 3 mice of each type).

Fig. 7.

ROS degradation increases ENaC activity in the PKC-α knockout lung. ENaC activity was recorded from cell-attached patches on isolated alveolar type II cells from wild-type mice or PKC-α knockout mice before and after addition of Tempol, a superoxide scavenger (pipette potential = 20 mV). A: addition of Tempol has little effect on ENaC in wild-type cells. B: addition of Tempol to isolated alveolar type II cells from PKC-α knockout mice resulted in a reversal of the observed decrease in ENaC activity compared with the wild-type. C: significant increase in the P_o of ENaC in the PKC-α knockout mice after the addition of Tempol (*P < 0.001 by paired t-test, n = 14), whereas there was no significant change in the wild-type mice. D: significant increase in the channel density of ENaC at the membrane in the knockout mice after the addition of Tempol (*P = 0.008), but there was no significant change seen in the wild-type animals (n = 14).

Fig. 8.

Inhibition of mitogen kinase phosphatase (MKP) in wild-type cells mimics the reduction in channel density in knockout animals. We examined the effect of the MKP1/3 inhibitor [(E)-2-Benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one] on ENaC activity in alveolar type II cells from wild-type and knockout animals. A and B: effect on channel density (measured as the number of measurable single channel levels in a patch). There is a significant reduction between untreated wild-type and MKP-inhibited wild-type (n = 4) and both knockout groups (indicated by an asterisk; n = 5). There is no significant difference between MKP-inhibited wild-type, untreated knockout, and MKP-inhibited knockout (determined by 1-way ANOVA). C and D: there appears to be no effect of MKP inhibition on P_o in either wild-type or knockout cells (paired t-test, n = 4 and 5, respectively).

Fig. 9.

Active PKC-δ is increased in the PKC-α knockout lung. Using Western blotting, we measured total PKC-δ in whole lung homogenates from PKC-α knockout and wild-type mice. To become active, the inactive form of PKC-δ must undergo modification. A: blot from alveolar type II cell lysates from wild-type and knockout animals (above). We determined that the amount of active PKC-δ, represented by the bottom band with a molecular weight around 50 kDa, is increased compared with the inactive form (*P < 0.01, n = 6). B: we repeated the experiment in the presence of instilled PKC-δ inhibitor to show that the inhibitor reduces the active form of PKC-δ (n = 6, difference in the ratio is not significant). We used Image J to quantify the blots. The program calculated the cumulative sum of the pixel values above the background for individual bands.

Fig. 10.

Inhibition of PKC-δ increases ENaC activity in the PKC-α knockout lung. ENaC activity was recorded from cell-attached patches on isolated alveolar type II cells from wild-type mice or PKC-α knockout mice, before and after addition of 1 μM a membrane-permeable, peptide pseudosubstrate inhibitor of PKC-δ (delta PKC, 8–17 from AnaSpec, Fremont, CA). A: there is little effect of a PKC-δ inhibitor on ENaC activity in cells from wild-type mice. B: addition of a PKC-δ inhibitor to isolated alveolar type II cells from PKC-α knockout mice resulted in a reversal of the observed decrease in ENaC activity compared with the wild-type. There was a significant increase in the P_o of ENaC in the PKC-α knockout mice after the addition of the PKC-δ inhibitor (*P = 0.009, n = 16), whereas there was no significant change in the wild-type mice (C). Also, there was a significant increase in the channel density of ENaC at the membrane in the knockout mice (*P = 0.003, n = 10), after the addition of the PKC-δ inhibitor, but there was no significant change seen in the wild-type animals (D).

Fig. 11.

Activation and inhibition of PKC in wild-type cells. A: ENaC activity recorded from paired, cell-attached patches on isolated alveolar type II cells from wild-type mice, before and after addition of a general PKC inhibitor, GF109203X (500 nM). The inhibitor produced a large increase in every cell (*P < 0.001 by paired t-test, n = 9). B: ENaC activity recorded from alveolar type II cells: untreated cells and treated with 100 nM phorbol myristoyl acetate (PMA) for 2 h before forming patches. ENaC activity was significantly inhibited in PMA-treated cells (*P < 0.001, n = 20 for both treated and untreated).

Fig. 12.

Schematic diagram of alveolar PKC signaling in wild-type and PKC-α knockout mice. A: wild-type lung. PKC-α is active, stimulating SOD, which breaks down ROS. In the absence of ROS, PKC-δ is inactive and does not phosphorylate MARCKS protein, which allows MARCKS to sequester phosphatidylinositol phosphate (PIP₂) near ENaC in the membrane, increasing ENaC P_o. In addition, ERK1/2 is not phosphorylated by PKC-δ, so that ENaC density at the membrane is increased attributable to decreased internalization. In addition, without high levels of superoxide in the lung, MKP is active, which dephosphorylates ERK1/2, leading to reduced apical expression of ENaC. B: in the knockout lung, in the absence of PKC-α, SOD is less active, leading to elevated ROS. ROS activates PKC-δ and inhibits MKP. The combination leads to increased ERK1/2 phosphorylation via PKC-δ and decreased dephosphorylation via MKP, which together activate ERK. ERK in turn phosphorylates ENaC, which promotes interaction with Nedd4–2, causing the ubiquitination and subsequent internalization of ENaC. ROS activation of PKC-δ promotes phosphorylation of MARCKS protein, which, when phosphorylated, leaves the membrane and does not sequester PIP₂ in proximity to ENaC. Without this interaction with PIP₂, ENaC P_o is decreased.