Mitochondrial PKCε and Mitochondrial ATP-Sensitive K⁺ Channel Copurify and Coreconstitute to Form a Functioning Signaling Module in Proteoliposomes

Chemicals and Reagents

The PKCε-specific peptides εV_1-2 (EAVSLKPT) and ψεRACK (HDAPIGYD) were synthesized with stated purity >98% by EZBiolab (Westfield, Ind.), according to the amino acid sequences described by Dorn et al.³ The PKCε inhibitor peptide control, a scrambled peptide to εV_1-2 (LSETKPAV), was from Calbiochem (San Diego, Calif.), and the anti-PKCε antibody (clone 21) was from BD Biosciences (San Jose, Calif). PP2A was from Calbiochem (San Diego, Calif) and exhibited a specific activity of 1800 U/mg as supplied. BODIPY-FL-glibenclamide and the potassium binding fluorescent indicator potassium-binding benzofuran isophthalate (PBFI) were from Molecular Probes (Eugene, Ore). All other chemicals were from Sigma-Aldrich Chemical Co (St. Louis, Mo).

Isolation of Mitochondria and Fractionation of Mitochondrial Proteins

Rat liver mitochondria (RLM) were used for these studies. RLM possess the same PKCε -dependent mitoK_ATP opening as heart and brain mitochondria,² and the yield of RLM is much greater because of the greater amount of starting material. Mitochondria were isolated and purified on a Percoll gradient exactly as previously described.² The mitochondrial protein extraction and fractionation followed previously described protocols,^4,5 with modifications. Mitochondria were solubilized at a protein concentration of 5 mg/mL in column buffer containing Triton X-100 (46.5 mmol/L), EDTA (1 mmol/L), β-mercaptoethanol (13 mmol/L), and Tris (50 mmol/L), pH 7.2 adjusted by HCl. Approximately 600 mg of the extracted proteins was loaded on 10 mL of DEAE-cellulose ion exchange column at a flow rate of 0.4 mL/min. The loading of the first 300 mg of protein was followed by washing the column with 2 column bed volumes of column buffer, containing Triton X-100 (3 mmol/L), EDTA (1 mmol/L), β-mercaptoethanol (13 mmol/L), and Tris (50 mmol/L), pH 7.2 adjusted by HCl. The remaining 300 mg of the protein was loaded and the column was washed for 10 hours at a flow rate of 0.1 mL/min with column buffer containing 100 mmol/L KCl. The column was further washed sequentially with column buffer containing 150, 250, and 500 mmol/L KCl, at least 2 column bed volumes each, at a flow rate of 0.4 mL/min. MitoK_ATP activity was found in the 250 mmol/L KCl fraction,⁴ referred to here as the “active fraction”.

Reconstitution and K⁺ Flux Measurements

Reconstitution of mitoK_ATP activity into PBFI-loaded liposomes was performed as described previously.⁵ Intraliposomal buffer contained PBFI (150 μmol/L) and tetraethylammonium (TEA⁺) salts of SO₄ (100 mmol/L), EDTA (1 mmol/L), and Hepes (25 mmol/L), pH 6.8. Kinetic studies were performed in external medium containing MgCl₂ (0.5 mmol/L) and K⁺ salts of SO₄ (80 mmol/L), and Hepes (25 mmol/L), pH 7.2. Electrophoretic K⁺ flux through mitoK_ATP was initiated by FCCP (0.25 μmol/L), which provides charge compensation. Fluorescence changes of the K⁺-sensitive probe PBFI were monitored using an SLM/Aminco 8000C fluorescence spectrophotometer (λ_ex/λ_em=345/485 nm), with fluorescence signals calibrated to K⁺ flux as previously described.⁶ Control experiments were performed using an ion exchange chromatography fraction that did not exhibit specific mitoK_ATP activity. None of the tested compounds had any effect on these K⁺ fluxes (data not shown). As is usual, we observed K⁺ flux in the presence of ATP (see Figure 3), which was about twice as high as background diffusive K⁺ flux (data not shown). As this level of flux is only observed in the fraction containing mitoK_ATP activity, we speculate that it is attributable to insertion of incomplete and unregulatable mitoK_ATP channels.

Western Blot Studies

Proteins from RLM (85 μg) and from the active fraction (3 μg to 6 μg) were precipitated using methanol-chloroform ref⁷ (with minor modifications) and resolved on 10% SDS-PAGE gels. Proteins separated on the gels were silver stained (Silver Stain Plus, Bio-Rad) or transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P transfer membrane, Millipore). After blocking with gelatin (25 g/L) in Tris-buffered saline, the PVDF mebranes were exposed to anti-PKCε antibody in a 1:500 dilution followed by alkaline phosphatase-conjugated secondary antibody (Immunoblot Assay kit goat anti-mouse IgG alkaline phosphatase, Bio-Rad). A colorimetric assay was used to visualize antigen-antibody reactions following manufacturer’s instructions.

BODIPY-FL-glibenclamide Labeling of the DEAE-Cellulose Fractions

Aliquots of each fraction eluted from the DEAE-cellulose column were incubated with BODIPY-FL-glibenclamide (100 nmol/L) in the presence or absence of unlabeled glibenclamide (1 μmol/L) for 10 minutes at room temperature. Adding a 10-fold excess of unlabeled glibenclamide is not expected to affect nonspecific (low-affinity) labeling, but it is expected to compete at specific (high-affinity) binding sites and to lower BODIPY-FL-glibenclamide labeling at those sites at least 10-fold. The samples were transferred to Costar 24-well ultra-low attachment cluster plates and UV-irradiated for 6 minutes on ice in a UVP CL-1000 Ultraviolet Crosslinker at 200 mJ/cm². To remove unbound BODIPY-FL-glibenclamide, the samples were precipitated using trichloroacetic acid and sodium deoxycholate followed by two 5-minute 400 μL acetone washes. The samples were dried and resuspended in TEA-EDTA (1 mmol/L), SDS (17.3 mmol/L), and Tris-HCl (50 mmol/L), pH 7.2. The fluorescence emission spectrum for each sample from 495 to 565 nm was collected using an SLM/Aminco 8000C spectrofluorometer, and analyzed for BODIPY-FL-glibenclamide fluorescence (λ_ex/λ_em=493/515 nm). For each fraction, the 2 emission spectra, with and without unlabeled glibenclamide, were integrated between 510 and 550 nm. Subtracting the former from the latter and expressing the difference as a ratio over the unlabeled glibenclamide control corrected for small differences in the protein concentration.

Copurification of Mitochondrial PKCε With MitoK_ATP

Our previous finding that activation of PKCε causes mitoK_ATP opening in heart, liver, and brain mitochondria² indicated that PKCε is constitutively localized in mitochondria. This was confirmed by immunodetection, as shown in Figure 1A. A clear signal at ≈90 kDa was observed in RLM (lane 1), which persisted after Percoll purification (lane 2). The abundance of mitoK_ATP is very low, and efficient regulation would seem to require colocalization of mitoK_ATP and PKCε. Accordingly, we investigated whether PKCε and mitoK_ATP copurify following detergent extraction and subsequent fractionation using ion exchange column chromatography. The immunoblot shown in lane 3 of Figure 1A was performed on the reconstitutively active fraction containing mitoK_ATP and demonstrates the presence of PKCε in this fraction. None of the other fractions from the ion exchange column exhibited a positive reaction with anti-PKCε antibodies (not shown). Essentially identical results were obtained using rat heart mitochondria (n=4, data not shown).

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Figure 1. Panel A, Immunoblot using anti-PKCε antibodies on rat liver mitochondria (lane 1); Percoll-purified mitochondria (lane 2); the reconstitutively active fraction (lane 3); and recombinant PKCε (lane 4). Lanes 1 and 2 contained 85 μg proteins, and lanes 3 and 4 contained 6 μg of proteins. Each result is representative of 3 to 4 independent experiments. Panel B, Silver-stained SDS-polyacrylamide gel containing 3 μg of proteins from the reconstitutively active fraction from rat liver mitochondria.

Figure 1B contains an SDS-PAGE gel of the reconstitutively active fraction. Numerous bands were observed on silver staining, as is typical of rat liver mitochondria. Figure 1B shows the band pattern we currently find in the fraction used for reconstitution experiments. The band pattern of the reconstitutively active fraction is similar but not identical to the patterns shown previously by Paucek et al,⁴ who used RLM, and by Bajgar et al,⁵ who used rat brain mitochondria. We attribute the differences in the observed protein band pattern to the differences in selected tissue and staining procedure, to the modifications in the protein fractionation protocol, which included broader salt step gradient, to a different flow rate of the ion exchange column, and to the fact that a much smaller aliquot was used for the gel in Figure 1B, so that minor bands were emphasized (see Materials and Methods).

To further confirm colocalization of PKCε and mitoK_ATP, we performed BODIPY-FL-glibenclamide labeling experiments⁵ on the column fractions, with the results shown in Figure 2. It can be seen that the fraction containing PKCε (“250” fraction) also exhibited specific labeling with BODIPY-FL-glibenclamide. High affinity glibenclamide binding is one of the ascribed properties of mitoK_ATP and these data provide strong evidence that PKCε copurifies with mitoK_ATP. We shall refer to this pool of PKCε as mitoPKCε.

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Figure 2. The bar graph is representing high affinity BODIPY-FL- glibenclamide binding in the 100, 150, 250, and 500 mmol/L KCl fractions from DEAE-cellulose ion exchange column. For each fraction, the fluorescence emission spectra in the presence or absence of unlabeled glibenclamide (1 μmol/L) were collected and integrated from 510 to 550 nm. The former was subtracted from the latter and the result is expressed as a ratio of the difference over the unlabeled glibenclamide control. These data depict the means±S.D. for 4 or more independent labeling experiments.

Functional Association of MitoPKCε With MitoK_ATP

We next performed experiments to examine whether the colocalized mitoPKCε and mitoK_ATP retained a functional association through detergent extraction, partial purification and reconstitution in liposomes. If this were so, the activation of endogenous PKCε should reverse ATP inhibition of K⁺ flux in proteoliposomes as it does in isolated mitochondria.² Indeed, this was found to be the case, as shown in Figure 3. In these experiments, K⁺ flux in the open state (Figure 3, no ATP) was inhibited by ATP (Figure 3, ATP), as previously shown.⁴ Addition of the phorbol ester PMA reversed ATP inhibition so that K⁺ flux returned to the control value (Figure 3A, ATP+PMA). Addition of the inactive 4α analogue of PMA did not reverse ATP inhibition (Figure 3A, ATP +4αPMA). The PKC inhibitor, chelerythrine, blocked the activating effect of PMA (Figure 3A, ATP+chel+PMA).

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Figure 3. Shown are traces reflecting electrophoretic K⁺ influx in proteoliposomes containing reconstituted mitoK_ATP as monitored by PBFI fluorescence. Panel A, Addition of 250 nmol/L FCCP caused electrophoretic K⁺ influx (no ATP) which was inhibited by 0.2 mmol/L ATP (ATP). The addition of 0.1 μmol/L PMA reversed the ATP inhibition (ATP+PMA). The effect of PMA was inhibited by 50 nmol/L chelerythrine (ATP+chel+PMA). Addition of 0.1 μmol/L of inactive phorbol ester was without effect (ATP+4αPMA). Panel B, Addition of 250 nmol/L FCCP caused electrophoretic K⁺ influx (no ATP) which was inhibited by 0.2 mmol/L ATP (ATP). The addition of 1 μmol/L ψεRACK reversed the ATP inhibition (ATP+ψεRACK). The effect of ψεRACK was prevented by 1 μmol/L εV_1-2 (ATP+εV_1-2+ ψεRACK). Addition of 1 μmol/L of scrambled peptide analog was without effect (ATP+εV_1-2-S+ψεRACK).

In a similar experiment, addition of the PKCε peptide agonist ψεRACK reversed the ATP inhibition (Figure 3B, ATP+ψεRACK). Addition of the PKCε-specific peptide antagonist εV_1-2 blocked the activating effect of ψεRACK (Figure 3B, ATP+εV_1-2+ψεRACK). The scrambled peptide analog to εV_1-2 did not block the effect of ψεRACK (Figure 3B, ATP+εV_1-2-S+ψεRACK) or the effect of PMA (data not shown). Ten-fold higher concentrations of the scrambled peptide analog were also without an effect (data not shown).

The results obtained from 3 or more independent reconstitutions examining the effects on K⁺ flux of various PKC agonists and antagonists are summarized in Figure 4A. The addition of the general PKC activators PMA and H₂O₂, as well as the specific PKCε peptide agonist ψεRACK, increased K⁺ fluxes in the presence of ATP nearly to the level of control. MitoK_ATP opening by PKCε activators was inhibited by both chelerythrine and the PKCε-specific peptide antagonist εV_1-2. MitoK_ATP opening by PKCε activators was also inhibited by the mitoK_ATP inhibitor 5-HD. These results show that PKCε and mitoK_ATP copurified and remained functionally and physically associated through purification and reconstitution.

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Figure 4. Panel A, The bars summarize experiments examining the effects of PKCε agonists on reconstituted mitoK_ATP in the presence of 0.2 mmol/L ATP. The agonists used were PMA (0.1 μmol/L), H₂O₂ (10 μmol/L), and ψεRACK (1 μmol/L). The half-maximal activation because of ψεRACK was ≈0.25 μmol/L (data not shown). Panel B, The bars summarize experiments comparing the effect of PKCε antagonists on K⁺ fluxes in the presence of 30 μmol/L diazoxide (Dzx) and 0.2 mmol/L ATP, and the FCCP-induced K⁺ fluxes in the absence of ATP. Experiments were performed as described in the legend to Figure 3. The concentrations of chelerythrine, εV_1-2 and 5-HD were 50 nmol/L, 1 μmol/L, and 0.5 mmol/L, respectively. The half-maximal inhibition by εV_1-2 was ≈0.3 μmol/L (data not shown). The data±S.D. were obtained from 3 or more independent reconstitutions. ATP-sensitive K⁺ flux was calculated as a ratio of rates corrected for the flux in the presence of ATP (ΔJ) and normalized for the maximal ATP-sensitive control rate (ΔJ_max).

We performed analogous experiments using the cardioprotective pharmacological opener diazoxide.⁸ The results of 3 independent reconstitutions are summarized in Figure 4B. As shown previously,⁹ ATP inhibition of K⁺ flux was reversed by diazoxide, and the diazoxide-opened channel was inhibited by 5-HD. An important point to note in Figure 4B is that the PKC antagonists, chelerythrine and εV_1-2, had no effect on the K⁺ flux induced by diazoxide. Additional control experiments were performed on the proteoliposomes, using the open state of mitoK_ATP in the absence of ATP, which should be insensitive to regulation. Figure 4B shows that none of the tested compounds had any significant effect on the FCCP-induced K⁺ fluxes observed in the absence of ATP.

Reversal of PKCε-Dependent MitoK_ATP Opening By PP2A

Our results are consistent with a mitoPKCε-dependent phosphorylation of mitoK_ATP or an intermediate protein that activates mitoK_ATP. The results in Figure 5 confirm that PKCε-dependent mitoK_ATP opening was mediated by a Ser/Thr kinase by demonstrating its reversal with PP2A, a Ser/Thr protein phosphatase. These experiments were performed by adding PMA, chelerythrine, and PP2A sequentially at 10s intervals. PMA reversed ATP inhibition of K⁺ flux, as shown in Figure 3. Addition of chelerythrine after PMA had no effect (Figure 5, PMA+chel), showing that chererythrine cannot block the effect of PKCε after phosphorylation has occurred. The subsequent addition of PP2A, 10 seconds before the addition of FCCP, reversed the activating effect of PMA (Figure 5, PMA+chel+PP2A). In the absence of PMA, PP2A had no effect on K⁺ flux (Figure 5, chel+PP2A), demonstrating that PP2A had no further effect on the ATP-inhibited K⁺ flux and implying that mitoK_ATP as purified is not significantly phosphorylated.

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Figure 5. Shown are traces reflecting electrophoretic K⁺ influx in proteoliposomes containing reconstituted mitoK_ATP as monitored by PBFI fluorescence. The electrophoretic K⁺ influx was initiated by the addition of 250 nmol/L FCCP. Addition of 0.2 mmol/L ATP was followed by the addition of 50 nmol/L PMA and, after 10 seconds, the addition of 50 nmol/L chelerythrine (ATP+PMA+chel). The addition of 22 ng/mL exogenous PP2A following the addition of chelerythrine 10 seconds before the addition of FCCP reversed the effect of PMA (ATP+PMA+ chel+PP2A). In the absence of PMA, chelerythrine and PP2A had no effect on the ATP-inhibited K⁺ flux (ATP+chel+PP2A).