AJP - Heart pressure measurements
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H322-H332, 2007. First published March 9, 2007; doi:10.1152/ajpheart.01035.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/H322    most recent
01035.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garg, V.
Right arrow Articles by Hu, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garg, V.
Right arrow Articles by Hu, K.

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

Vivek Garg and Keli Hu

Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio

Submitted 21 September 2006 ; accepted in final form 5 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ATP-sensitive K+ (KATP) channels in both sarcolemmal (sarcKATP) and mitochondrial inner membrane (mitoKATP) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcKATP contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoKATP remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing KATP into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing KATP channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing KATP channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional KATP channels in mitochondria was significantly increased by PMA, as shown by KATP-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing KATP channels was blocked by a selective PKC{epsilon} inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the KATP channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKC{epsilon}. PKC isoform-regulated mitochondrial import of KATP channels may have significant implication in cardioprotection of IPC.

ischemic preconditioning; mitochondria; Kir6.2

ACTIVATION of ATP-sensitive potassium (KATP) channels could protect the heart under metabolic stress (27, 43–45). Importantly, the modulation of KATP channels accounts for the ability of brief ischemia and reperfusion to protect the heart against infarction induced by subsequent prolonged ischemia, a phenomenon known as ischemic preconditioning (IPC) (20, 26, 43, 47). Not only do molecularly defined cell surface (sarcolemmal) KATP (sarcKATP) channels in the heart activate on metabolic stress and cause action potential shortening and less energy consumption (30, 45, 60), KATP channels in the mitochondrial inner membrane (mitoKATP channels) are also implicated in cardioprotection during ischemia (22, 26, 47). However, despite immense interest and serious attempts to clone mitochondrial KATP channels, their molecular nature remains elusive.

The KATP channels in the sarcolemma membrane, first described in the heart by Noma (46) in 1983, are widely distributed in many tissues and cell types including pancreatic beta-cell, neuron, skeletal muscle, and smooth muscle (6, 7). The major function of the KATP channel is to couple the cell metabolic state to its membrane potential by sensing changes in intracellular adenine nucleotide concentration (4). The sarcolemmal KATP channels are heterooctamers consisting of two structurally unrelated proteins (1, 2, 8, 52, 54): four pore-lining subunits that belong to the Kir6 subfamily of inwardly rectifying potassium channels and four regulatory subunit sulfonylurea receptors (SUR) of the ATP-binding cassette (ABC) superfamily. It is currently known that the cardiac sarcKATP channel is composed of an octomeric complex of two types of subunits, the Kir6.2 and the SUR2A subunit.

Whereas the mitoKATP channel has been characterized pharmacologically (14, 23), its molecular nature remains unknown. Whether Kir6.2 contributes to mitoKATP channels is under debate; the subunit has been found in heart mitochondria (15, 36, 56, 74). We believe that much of the controversy or difficulty regarding the existence of Kir6.2-containing KATP channels in mitochondria may be due to the low level of basal KATP channels present in mitochondria. All these studies concerning the existence of Kir6.2-containing KATP in mitochondria were conducted under basal conditions that do not involve IPC or its signaling molecule(s), a critical step that may upregulate protein transport to mitochondria and thus confer cardioprotection (10).

Although KATP channel regulation and implication in cardioprotection have been studied by many groups for years, no study has ever explored the trafficking aspect of KATP function in mitochondria. The present study utilizing KATP-deficient COS-7 cells represents a novel approach to elucidation of the molecular basis of the KATP channels in mitochondria and their regulatory mechanism. We report the novel finding that a protein kinase C (PKC) isozyme, PKC{epsilon}, promotes mitochondrial import of Kir6.2-containing KATP channels from cytosol, assessed with a combination of immunofluorescence microscopy, Western blot, fluorescence resonance energy transfer (FRET) analysis, and mitochondrial functional studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DNA constructs. The hemagglutinin (HA) epitope was introduced into mouse Kir6.2 cDNA by sequential overlap extension polymerase chain reaction (PCR) as described previously (73). The epitope was inserted at position 102 of Kir6.2. The 11 amino acids (98GDLYAYMEKGIT99) were inserted into Kir6.2 before the HA epitope. Mammalian expression constructs were either in pcDNA3 (Invitrogen) or pEGFP/pECFP/pEYFP (Clontech). Mito-cyan fluorescent protein (CFP) or Mito-yellow fluorescent protein (YFP) was constructed by subcloning the mitochondrial presequence (the NH2-terminal 12 amino acids of the presequence) from subunit IV of cytochrome-c oxidase into the pEGFP-N1, pECFP-N1, or pEYFP-N1 vector (Clontech). To generate green fluorescent protein (GFP), YFP, or CFP fusion proteins, full-length Kir6.2 was amplified by PCR and ligated into pEGFP-N1, pECFP-N1, or pEYFP-N1. All constructs were verified by DNA sequencing.

Cell culture and transfection. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-F-12 supplemented with 10% fetal bovine serum, 2 mM glutamine, and penicillin-streptomycin (30). Cells were grown on coverslips or petri dishes depending on experimental purpose. Cells were transfected with HA-tagged (Kir6.2-HA) or GFP-fused (Kir6.2-GFP) Kir6.2 and SUR2A (pCMV) KATP channel subunits or vectors alone by using FuGENE6 (30). Two to three days later, the cells were treated with phorbol 12-myristate 13-acetate (PMA, 100 nM), PMA plus the PKC inhibitor chelerythrine (10 µM), or PMA plus the selective PKC{epsilon} inhibitor peptide myristoylated PKC{epsilon} V1-2 (PKC{epsilon} V1-2, 10–20 µM) for 30–60 min before the experiments. 4{alpha}-PMA (100 nM) was used as negative control.

Isolation of cardiomyocytes. Adult ventricular myocytes were isolated from Wistar rats (250–300 g) by enzymatic dissociation (29, 31). The animal procedure was approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee. In brief, hearts were excised and retrogradely perfused via the aorta with oxygenated (100% O2) Tyrode solution containing (mM) 126 NaCl, 5.4 KCl, 1.0 CaCl2, 1.0 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucose at 37°C. The perfusate was then changed to a Tyrode solution that was nominally Ca2+ free but otherwise had the same composition. The hearts were perfused with the same solution containing collagenase for 20 min. The digested cell suspension was gently centrifuged, and the pellet was resuspended in culture medium 199 with 2% fetal bovine serum and 1% penicillin-streptomycin at 37°C. Cardiomyocytes were placed in 100-mm petri dishes and incubated at 37°C in a humidified 5% CO2-95% air mix. Cardiomyocytes were subjected to various pretreatments before mitochondrial isolation.

Preparation of mitochondria and mitoplasts. Mitochondrial fractions and total cell homogenate were prepared from cultured COS-7 cells transiently transfected with Kir6.2 and SUR2A, isolated by differential centrifugation, and further purified by 30% Percoll ultracentrifugation. Cells transfected with vector alone were always used as a negative control. For intact mitochondria or mitoplast colocalization experiments, COS-7 cells were transfected with Kir6.2-GFP and SUR2A. Both cardiomyocytes and COS-7 cells were stained with the mitochondrial marker MitoTracker (250 nM) for 15 min before fractionation. Cells after treatment were collected in an ice-cold homogenizing buffer containing (mM) 250 sucrose, 5 HEPES, and 5 EDTA, with proteinase inhibitor cocktail. Two 15-s homogenization cycles were performed on ice. The homogenate was centrifuged at 1,000 g for 10 min to remove nuclei and debris. The supernatant was centrifuged at 8,500 g for 20 min. The pellet containing the mitochondrial fraction was resuspended in the homogenizing buffer and further centrifuged at 8,500 g for 20 min. The washed mitochondria were then resuspended. For Percoll purification, the crude mitochondrial suspension (0.5 ml) was laid on the top of 10 ml of a solution containing 30% Percoll, 0.25 M sucrose, 1 mM EDTA, and 10 mM HEPES (pH 7.4). A self-generating Percoll gradient was developed by centrifugation at 95,000 g for 30 min at 4°C. The mitochondrial band was collected with a Pasteur pipette and washed in the homogenizing buffer. Mitoplasts were prepared from intact mitochondria by osmotic shock (55). Briefly, mitochondria were subjected to osmotic shock for 5 min in hypotonic solution containing (mM) 5 sucrose, 5 HEPES, and 1 EGTA. Mitoplasts were sedimented from this solution by centrifugation at 3,920 g for 5 min and resuspended in hypertonic solution containing (mM) 750 KCl, 100 HEPES, and 1 EGTA (pH 7.2 with KOH).

Western blotting. Immunoblot analysis was carried out for the mitochondrial fraction and total cell homogenate from COS-7 cells expressing Kir6.2 and SUR2A after treatment. The purity of mitochondria was evaluated with antibody against the mitochondrial marker protein prohibitin, the plasma membrane marker Na+-K+-ATPase, and the endoplasmic reticulum marker calreticulin to ensure that there was no significant contamination of other membrane fractions in mitochondria. Mitochondria and total cell homogenate were denatured in a sample buffer, electrophoresed on 8–10% SDS-polyacrylamide gels (37), and transferred onto nitrocellulose membranes (61). The transferred blots were blocked with 5% nonfat milk in Tris-buffered saline (TBST; 150 mM NaCl, 20 mM Tris·HCl, 0.05% Tween 20, pH 7.4) and incubated for 1 h at room temperature with the antibodies against HA epitope (Roche) or Kir6.2 or SUR2A (Santa Cruz Biotechnology) in TBST. After being washed, the blots were reacted with peroxidase-conjugated secondary antibodies (Jackson Immunuolabs) for 45 min and developed with an enhanced chemiluminescence detection system. The specificity of antibodies for Kir6.2 and SUR2A was tested by preincubation of antibodies with their antigen peptides and/or confirmed in nontransfected COS-7 cells or transfected cells with different KATP subunit. Equal loading was confirmed by staining with Ponceau S or using antibodies against {alpha}-tubulin.

Immunofluorescence microscopy. Mitochondria or mitoplasts from cells after treatments were fixed with 4% formaldehyde in PBS for 30 min, blocked, permeabilized in 5% goat serum in PBS with 0.1% Triton X-100 (30 min), and labeled with primary antibody against HA tag or Kir6.2 for 2 h. Mitoplasts isolated from COS-7 cells transfected with Kir6.2-GFP/SUR2A were fixed only. Cells were washed three times and labeled with fluorescence-conjugated secondary antibody for 1 h (30). We used anti-Kir6.2 or anti-HA primary antibody for labeling KATP channel subunit Kir6.2 and the mitochondrion-specific stain MitoTracker (250 nM) for labeling mitochondria. The cells were incubated with MitoTracker for 15 min before isolation of mitochondria/mitoplasts. Immunofluorescence was visualized with a fluorescent microscope (Nikon) or a confocal scanning laser microscope (Zeiss).

FRET measurements. COS-7 cells were transfected with Kir6.2-CFP/SUR2A or Mito-YFP or cotransfected with both Kir6.2-CFP/SUR2A and Mito-YFP. Images were acquired sequentially through CFP, YFP, and FRET filter channels. Filter sets used were the donor CFP, the acceptor YFP, and FRET. A background value was determined from a region in each image without any cells. The background value was subtracted from the raw images before FRET calculations were carried out. Corrected FRET (FRETC) was calculated for entire images or selected regions of images, such as individual mitochondria, by using the equation FRETC = FRET – (0.5 x CFP) – (0.3 x YFP), where FRET, CFP, and YFP correspond to background-subtracted images of cells coexpressing CFP and YFP acquired through the FRET, CFP, and YFP channels, respectively. The 0.5 and 0.3 values are the fractions of bleed through of CFP and YFP fluorescence, respectively, estimated from cells expressing either CFP or YFP fusion proteins. Mean FRETC values were calculated from mean fluorescence intensities for each selected subregion. FRETC images are presented as a quantitative pseudocolor image. Data were analyzed with ImagePro and MetaMorph.

Mitochondrial membrane potential. The changes in mitochondrial membrane potential ({Delta}{Psi}m) were monitored with the dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) (51). Cells were treated with 4{alpha}-PMA, PMA, PMA plus chelerythrine, or PMA plus PKC{epsilon} V1-2 and then stained with JC-1 (5 µM) at 37°C for 15 min and rinsed three times with Tyrode solution. The mitoKATP opener diazoxide or diazoxide plus the mitoKATP inhibitor 5-hydroxydecanoic acid (5-HD) was added 20 min before the measurement. The observations were made with a fluorescence microscope. Solution changes in this protocol were made by aspirating and replacing the contents of the recording chamber. One hundred areas were selected from each image, and the average intensity for each region was quantified by MetaMorph. The ratio of JC-1 aggregate (red fluorescence) to monomer (green fluorescence) intensity for each region was calculated after background subtraction. A decrease in this ratio was interpreted as decrease of {Delta}{Psi}m, whereas an increase in the ratio was interpreted as gain in {Delta}{Psi}m (62). Cells lacking red fluorescence were considered severely damaged and were excluded from analysis.

Statistics. Group data are presented as means ± SE. Unpaired t-test was used to compare between groups. Multiple group means were compared by ANOVA followed by post hoc least significant difference test. Differences with a two-tailed P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of Kir6.2 in isolated mitoplasts. To determine whether PKC induces import of cardiac KATP to mitochondria, we took advantage of the COS-7 cell line, which lacks native KATP channels (33), to deliver genes encoding cardiac KATP channel subunits Kir6.2 and SUR2A. We first examined whether the PKC activator PMA promotes the mitochondrial import of recombinant cardiac KATP channels. 4{alpha}-PMA was used as a negative control. To avoid colocalization of Kir6.2 with the mitochondrial marker MitoTracker due to close apposition between Kir6.2-containing vesicles and mitochondria, we performed colocalization experiments in mitoplasts (devoid of outer membrane) freshly isolated from COS-7 cells transfected with SUR2A and Kir6.2 with GFP fused to its COOH terminus. The cells were pretreated with 4{alpha}-PMA, PMA, or PMA plus PKC{epsilon} V1-2 for 60 min and were stained with MitoTracker before isolation of mitochondria. Mitoplasts were obtained by subjecting intact mitochondria to hypotonic shock (see MATERIALS AND METHODS for details). About 20–30 images in the same treatment group (2 or 3 coverslips) from each experiment were analyzed. Three independent experiments were conducted. Without PMA (100 nM, data not shown) or with 4{alpha}-PMA (100 nM) treatment, only a small portion of fluorescent Kir6.2 was localized in mitoplasts that were stained with MitoTracker. PMA treatment significantly increased mitoplast localization of Kir6.2 (Fig. 1). Quantification analysis revealed that the activation of PKC by PMA increased the number of Kir6.2-positive mitoplasts by ~100% (Fig. 1C; 205.78 ± 8.84% vs. 4{alpha}-PMA, P < 0.01). To identify which PKC isozyme is involved in PKC-induced increase in mitochondrial localization of Kir6.2, we studied the effect of the selective PKC{epsilon} peptide inhibitor PKC{epsilon} V1-2. PKC{epsilon} has been shown to modulate KATP channels (32) and is linked to IPC (50). As expected, the effect of PMA on mitochondrial localization of Kir6.2 was completely prevented by PKC{epsilon} V1-2 (10 µM; 110.39 ± 10.33%). These results indicate that Kir6.2-containing KATP channels are localized in mitochondria at low levels under normal conditions and can be enhanced by the activation of PKC isozyme PKC{epsilon}.


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 1. Immunofluorescence microscopy of mitochondrial localization of Kir6.2 in mitoplasts isolated from COS-7 cells. A: transmitted and fluorescent images of a mitoplast (mitochondria devoid of outer membrane) labeled with the mitochondrial marker MitoTracker. Scale bar: 3 µm. B: COS-7 cells were transfected with sulfonylurea receptor (SUR)2A/Kir6.2-green fluorescent protein (GFP), and mitoplasts were prepared after COS-7 cells were treated with agents as indicated. Results are representative of 3 independent experiments. About 20–30 images in the same treatment group (2 or 3 coverslips) from each experiment were analyzed. Scale bar: 5 µm. PMA, phorbol 12-myristate 13-acetate; PKC{epsilon}, protein kinase C isoform {epsilon}. C: % of mitoplasts (MitoTracker positive) with Kir6.2-positive fluorescence are shown (data were normalized to the control without treatment). **P < 0.01 vs. 4{alpha}-PMA.

 
FRET analysis of Kir6.2 in mitochondrial inner membrane. Although our observation that Kir6.2 is localized in mitoplasts indicates that Kir6.2 is present in mitochondrial inner membrane, there is a possibility that part of the outer membranes of mitochondria may still be attached to mitoplasts. To further define whether KATP channels are indeed localized in mitochondrial inner membrane, we tested in COS-7 cells whether CFP fused to the COOH terminus of Kir6.2 is sufficiently close to Mito-YFP that is targeted into the mitochondrial matrix to yield FRET (24). If KATP channels are in the outer membrane of mitochondria, the fluorescent protein fused to the cytoplasmic domain of Kir6.2 would be exposed to the cytosol. If these channels actually reside in the mitochondrial inner membrane, their cytoplasmic domains should be exposed to the matrix enclosed by the mitochondrial inner membrane—the equivalent of the cytosol for these endosymbiotic organelles. As shown in Fig. 2, FRETC was minimal in all regions of cells that were not stimulated with PMA (Fig. 2A), indicating that there was no significant amount of Kir6.2 localized in mitochondria. In particular, FRET signals were not observed in the mitochondria enriched with Mito-YFP or in the Golgi area containing a large amount of Kir6.2-CFP. This validates the accuracy of our method of correction for the non-FRET component of the FRET images. Incubation of cells with PMA at 37°C for 30–60 min led to rapid import of Kir6.2 protein into mitochondria and exhibited FRET signals due to energy transfer from CFP at the COOH terminus of Kir6.2 to YFP in the mitochondrial matrix. To compare FRET efficiencies, the FRETC was normalized to the intensity of Kir6.2-CFP after background subtraction. The FRET signal between Mito-CFP and Mito-YFP was comparable to that between Kir6.2-CFP and Mito-YFP after PMA treatment for 60 min, indicating that by then the KATP channels targeted to the mitochondria had fully exposed the CFP at the COOH terminus of Kir6.2 to the matrix. Similar FRET signals were observed in cells expressing Mito-YFP, SUR2A, and Kir6.2 with CFP fused to its NH2 terminus (data not shown). For dipole-dipole coupling to induce energy transfer from donor to acceptor, the fluorophore at the NH2 or COOH terminus of Kir6.2 must be within 6–10 nm of the fluorophore within the mitochondrial matrix (24, 48). These findings strongly suggest that PKC activation causes insertion of Kir6.2-containing KATP channels into the mitochondrial inner membrane.


Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 2. A: Proximity of Kir6.2-cyan fluorescent protein (CFP) (left) to Mito-yellow fluorescent protein (YFP) in mitochondrial matrix (center) yielded fluorescence resonance energy transfer (FRET) signals (right, in quantitative pseudocolor) in cells treated with PMA (100 nM) for 60 min (bottom) but not in cells without PMA treatment (top). FRETC, corrected FRET. B: FRET values were normalized by dividing FRETC by the mean intensity of CFP after background subtraction. The FRET signal between Mito-YFP and Mito-CFP provides calibration for a strong signal produced by 2 matrix fluorescent proteins.

 
Alterations of Kir6.2 protein in mitochondria. To assess the effect of PKC activation on Kir6.2 protein level, immunoblot analysis was carried out for Percoll-purified mitochondria fraction and total cell homogenate prepared from COS-7 cells treated with 4{alpha}-PMA, PMA, PMA plus chelerythrine, or PMA plus PKC{epsilon} V1-2 for 60 min. Enrichment of mitochondria was established by labeling with anti-prohibitin, a molecular marker of mitochondria. With anti-HA antibody, which is known as a very good antibody, a single band at 37 kDa was detected in the mitochondrial fraction as well as total cell homogenate (Fig. 3, A and B). Anti-HA antibody did not show any nonspecific band in our study. Interestingly, the band in the mitochondrial fraction from COS-7 cells treated with PMA for 60 min exhibited a more intense signal than that from COS-7 cells without PMA treatment or with 4{alpha}-PMA treatment. The PMA effect was significantly eliminated not only by the general PKC inhibitor chelerythrine (10 µM) but also by the selective PKC{epsilon} inhibitor peptide PKC{epsilon} V1-2 (10 µM). However, there was no difference in the Kir6.2 band in total cell homogenate between cells treated with 4{alpha}-PMA and PMA. No Kir6.2 band was detected from cells without transfection. Similar results were obtained with antibody against Kir6.2 (data not shown). Figure 3B shows average Kir6.2 band intensity normalized to control from three independent experiments. PMA induces almost 100% increase in Kir6.2 protein level in the mitochondrial fraction compared with that in the control group (216.11 ± 20.29%), which is consistent with the data in mitochondrial localization of Kir6.2, but has no effect on total cellular Kir6.2 protein level. When we tried a similar experiment with anti-SUR2A antibody, there was a single faint band of SUR2A at a molecular mass of 150 kDa in the mitochondrial fraction under control conditions, which was not affected by PMA pretreatment (Fig. 3C). This result indicates that only a minimum amount of SUR2A exists in mitochondria, if there is any. However, we did not detect SUR2A with antibody against SUR2, which is similar to the report by Lacza et al. (36) showing that SUR2 was not present in mitochondria with the same antibody (purchased from the same company). This discrepancy is likely due to the differential sensitivities of two different antibodies. The specificity of anti-SUR2A antibody was confirmed in nontransfected COS-7 cells and COS-7 cells transfected with SUR2B showing no cross-reactivity with SUR2B. We noted that the anti-SUR2A antibody is very specific and more sensitive than the anti-SUR2 antibody. These results suggest that Kir6.2 is present in mitochondria and PKC{epsilon} activation by PMA further increases Kir6.2 protein level in mitochondria. This increase may not be due to increased protein synthesis for Kir6.2 since Kir6.2 in the total cellular fraction was not changed after PMA treatment.


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. Immunoblot analysis of Kir6.2 protein in mitochondria. COS-7 cells were transfected with Kir6.2-hemagglutinin (HA)/SUR2A. A: total cell homogenate (cellular fraction, CF) and mitochondrial fraction (MF) of COS-7 cells were prepared after cells were treated with 4{alpha}-PMA, PMA, PMA + chelerythrine (Che), or PMA + PKC{epsilon} V1-2. These proteins were separated on an 8–10% SDS-polyacrylamide gel and immunoblotted with antibodies against HA (top) and prohibitin (bottom). Thirty micrograms of protein for each lane was loaded for cellular fraction, whereas 20 µg of protein was loaded for mitochondrial fraction analysis. B: signal intensity of Kir6.2 in cellular and mitochondrial fractions was quantified as values normalized to control from 3 independent experiments with Image J software. C: similar Western blot analysis is shown for SUR2A in mitochondrial fraction with antibody against SUR2A.

 
Kir6.2-dependent changes in mitochondrial membrane potential. To determine whether Kir6.2-containing KATP channels in mitochondria are functional, we measured the changes in {Delta}{Psi}m in intact COS-7 cells transfected with Kir6.2 and SUR2A, using the potential-sensitive dye JC-1. About 100 regions were selected from each image, which contains 10–20 cells, and 5 images were taken from each well. Three independent experiments were conducted, and each experiment had triplicate wells of the same treatment. The average intensity for each image was quantified after background subtraction. The green fluorescence of monomer indicates low potential (depolarized), and the red fluorescence of J-aggregate indicates high potential (hyperpolarized). Since KATP channels are usually not active because of inhibition by matrix ATP, the mitochondrion-selective KATP opener diazoxide was used to activate KATP in mitochondria. Diazoxide has been shown to confer the cardioprotection of IPC by selectively activating mitoKATP channels (22, 47, 67). If Kir6.2/SUR2A-containing KATP channels are localized in mitochondrial inner membrane and involved in the protection of IPC, this channel is expected to be sensitive to activation by diazoxide. The KATP-dependent changes were verified by the mitochondrial KATP-selective inhibitor 5-HD. In the absence of diazoxide, the JC-1 ratio (J-aggregate/monomer) in cells pretreated with PMA for 60 min was not significantly different from the control cells pretreated with 4{alpha}-PMA (Fig. 4B). However, diazoxide (100 µM), within 15 min of application, decreased the JC-1 ratio significantly in PMA-treated cells (potential decreased, green fluorescence increased; Fig. 4A) compared with that in 4{alpha}-PMA-pretreated cells with diazoxide (Fig. 4; 53.04 ± 10.22% vs. 97.87 ± 9.24%, PMA+diazoxide vs. 4{alpha}-PMA+diazoxide, P < 0.01). When diazoxide was added to the solution 5 min after the application of 5-HD, the diazoxide-induced decrease in {Delta}{Psi}m in cells treated with PMA was inhibited not only by 5-HD (500 µM; 83.79 ± 6%) but also by pretreatment with PKC{epsilon} V1-2 (20 µM; 82.61 ± 4.56%). Either 5-HD application or PKC{epsilon} V1-2 pretreatment alone did not have any effect on {Delta}{Psi}m (data not shown). These data show that {Delta}{Psi}m in cells pretreated with PMA was not different from that in cells with 4{alpha}-PMA but could be decreased by activation of KATP channels in mitochondria with the mitoKATP opener diazoxide after PMA pretreatment, an effect that was inhibited by the mitoKATP inhibitor 5-HD. Furthermore, the diazoxide effect in PMA-treated cells was not seen in nontransfected COS-7 cells, indicating that the effect of diazoxide on {Delta}{Psi}m was due to activation of Kir6.2-containing KATP channels in mitochondrial inner membrane. The finding that diazoxide induces KATP-dependent depolarization of {Delta}{Psi}m after activation of PKC{epsilon} by PMA pretreatment but not in the control group with 4{alpha}-PMA treatment suggests that K+ influx caused by diazoxide under basal conditions may not be sufficient to cause significant changes in {Delta}{Psi}m under our experimental conditions. This result does not exclude the possibility that other mitochondrial functions may be altered by diazoxide under normal conditions or in isolated mitochondria where no translocation occurs. Furthermore, the observation that PMA pretreatment alone did not change {Delta}{Psi}m suggests that KATP channels in mitochondrial inner membrane are normally inhibited by matrix ATP even though PMA may increase the number of functional Kir6.2-containing KATP channels in mitochondria. It should be noted that PMA was used only for pretreatment and was washed out before measurement of {Delta}{Psi}m with diazoxide.


Figure 4
View larger version (52K):
[in this window]
[in a new window]

  		Fig. 4. PMA-induced KATP-dependent changes in mitochondrial membrane potential ({Delta}{Psi}m). A: COS-7 cells transfected with SUR2A/Kir6.2-HA were incubated with mitochondrial potential-sensitive dye JC-1 (5 µM) for 15 min after various pretreatments as indicated. Diaz, diazoxide. Scale bar: 20 µm. B: {Delta}{Psi}m expressed as a ratio of J-aggregate to monomer fluorescence in different treatments. Three independent experiments were conducted, and each experiment had triplicates of the same treatment where ~15 images were collected. The average fluorescent intensity for each image was calculated after background subtraction. Data from different treatment groups were normalized to the control group without PMA treatment. **P <0.01 vs. 4{alpha}-PMA.

 
Localization of Kir6.2 in isolated mitochondria from cardiomyocytes. To exclude the possibility that overexpression of recombinant KATP channels contributes to our findings, we performed similar colocalization experiments in isolated mitochondria from rat adult cardiomyocytes. Because of very tightly packed myofibrils around mitochondria and other organelles in cardiomyocytes, it is very difficult to study subcellular localization of membrane proteins in isolated cardiomyocytes. We therefore performed colocalization experiments in isolated mitochondria. Cardiomyocytes were subjected to pretreatment with 4{alpha}-PMA, PMA, or PMA plus PKC{epsilon} V1-2 for 60 min before mitochondrial isolation. To avoid bias in our analysis, 30 images were randomly collected from the triplicates of the same treatment in each independent experiment. Two to five independent experiments depending on treatments were conducted, and ~1,000 mitochondria were counted for statistical analysis. Anti-Kir6.2 antibody was used to detect native Kir6.2 protein. As Fig. 5A shows, PMA increased mitochondrial localization of Kir6.2, as indicated by increased yellow staining in the merged image. The pretreatment of PMA alone with PKC{epsilon} V1-2 blocked the PMA effect. Quantitative analysis by normalizing Kir6.2-positive mitochondria to the control without PMA treatment showed that PMA significantly increased the number of Kir6.2-positive mitochondria (Fig. 5B; 240.31 ± 13.6% vs. 4{alpha}-PMA, P < 0.01). This PMA effect was almost completely prevented by pretreatment with the PKC inhibitor chelerythrine (10 µM; data not shown) as well as the selective PKC{epsilon} inhibitor peptide PKC{epsilon} V1-2 (10 µM; 120.02 ± 9.77% vs. PMA group, P < 0.01). These results are consistent with our observation in COS-7 cells transiently transfected with Kir6.2/SUR2A and support the scenario that Kir6.2-containing KATP channels are localized in mitochondria and this mitochondrial localization was further enhanced by PKC activation.


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 5. Immunofluorescence microscopy of mitochondrial localization of Kir6.2 in mitochondria isolated from rat adult cardiomyocytes. A: fluorescent images of mitochondria with Kir6.2 labeled with anti-Kir6.2 antibody. The intact mitochondria were prepared after treatment of rat cardiomyocytes with agents as indicated. Results are representative of 2–5 independent experiments depending on the treatments, and each experiment had triplicates of the same treatment with 30 images taken. Scale bar: 10 µm. B: normalized % of mitochondria with Kir6.2-positive fluorescence over control. **P < 0.01 vs. 4{alpha}-PMA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study points to a novel trafficking mechanism for dynamic regulation of KATP channel number: PKC-induced import of KATP channels into mitochondria. We show that the KATP channel pore-forming subunit Kir6.2 is localized in mitochondria, using both cardiomyocytes and KATP channel-deficient COS-7 cells transiently transfected with Kir6.2 and SUR2A. Most importantly, we demonstrate that activation of PKC isozyme PKC{epsilon} significantly increases the number of functional Kir6.2-containing KATP channels in mitochondrial inner membrane, possibly by promoting import of Kir6.2 to mitochondria from cytosol. One likely scenario is that KATP channels are simultaneously targeted to not only cell surface but also mitochondrial inner membrane after their synthesis in the cytosol and PKC activation enhances the import of these channels to mitochondria. The specific pharmacological and biophysical properties of KATP channels at the plasma membrane and mitochondrial inner membrane may differ depending on the environment in which they reside and the regulatory factors with which they are associated (16).

The function of ion channels depends critically on not only channel activity but also the number of functional channels. Our observation that activation of PKC{epsilon} by PMA increases mitochondrial localization of Kir6.2-containing KATP channels points out a novel trafficking mechanism in regulation of KATP channel function. However, this finding does not exclude the possibility that PKC may directly interact with the channel. Actually, PMA has been shown to activate cell surface Kir6.2/SUR2A-containing KATP channels (29, 40) as well as mitoKATP channels (32). Under our experimental conditions, PMA was washed out before we tested diazoxide-induced changes in {Delta}{Psi}m. If there is any residue of PMA, it may not be enough to activate KATP channels and cause alterations in {Delta}{Psi}m. The present study mainly attempted to address whether KATP channels translocated by PKC activation are functional.

Our data and that of others (15, 36, 56, 74) show that there is a basal level of Kir6.2-containing KATP channels in mitochondria. It is therefore likely that K+ channel openers or stimulators such as PMA may directly activate these channels under normal conditions or in isolated mitochondria where no translocation occurs. In the present study, diazoxide alone did not change {Delta}{Psi}m in control cells without PMA or pretreated with 4{alpha}-PMA. One explanation is that K+ influx induced by diazoxide in COS-7 cells may not be high enough to cause significant changes in {Delta}{Psi}m but may produce changes in other mitochondrial functions that we did not measure, such as matrix volume. On the other hand, our observation that diazoxide caused changes in {Delta}{Psi}m after PMA treatment indicates that the KATP channels translocated by PMA are functional by increasing K+ influx. However, our data do not imply that the same functional consequence in mitochondria ({Delta}{Psi}m) caused by diazoxide-induced opening of KATP channels would occur in native cardiomyocytes. We employed COS-7 cells with expressed recombinant KATP channels, which may be different from cardiomyocytes in terms of the extent of K+ influx. We therefore do not believe that our data conflict with that of others showing no significant changes in {Delta}{Psi}m by diazoxide-induced opening of mitoKATP (14, 35) from isolated heart mitochondria. Nevertheless, the purpose of the JC-1 experiment was not to define ultimate changes in mitochondrial function by diazoxide but rather to study whether the translocated Kir6.2-containing KATP channels are functional. Diazoxide was used as a tool to activate KATP channels since KATP channels are normally inactive.

Although it is generally assumed that the Kir6.2/SUR2A-containing KATP channel is localized exclusively at the sarcolemma, there is evidence that Kir6.2 or SUR2A is present in mitochondria. Several groups have reported that Kir6.2 has been found more or less in isolated heart mitochondria (15, 36, 56, 74). One study has reported that Kir6.2 is not part of the components of mitoKATP channels in the rabbit heart (53). The discrepancy may be due to the relatively low level of Kir6.2-containing KATP channels in mitochondria under normal conditions. The technical difficulties in dissecting a small amount of membrane-targeted proteins localized in mitochondria with conventional immunofluorescence microscopy may contribute to the inconsistency, especially in adult cardiomyocytes with tightly packed myofibrils and suborganelles. Differential sensitivity of antibodies against individual KATP subunits may also likely be a reason for the discrepancy. With regard to the SUR subunit, less is known about its presence in mitochondria because of the difficulty in getting antibody specific to SUR2A or SUR2B. We tested anti-SUR2A antibody, recently available commercially, and found a faint band of SUR2A present in mitochondria of COS-7 cells by Western blot, which is consistent with some reports (15, 56). Similar to another study (36), we did not detect a SUR2 band with anti-SUR2 antibody in mitochondria. Because of the lack of series of studies with different approaches, we cannot make a conclusion at the present time that SUR2A is indeed localized in mitochondria.

The contribution of Kir6.1 to mitoKATP channels is also under debate (42, 57). The Kir6.1/SUR2B-based channels do not exhibit sensitivity to the inhibition by intracellular ATP (70), whereas the mitoKATP channels recorded in mitochondria inner membrane from liver and reconstituted lipid bilayer from purified heart mitochondria are very sensitive to ATP, a characteristic feature of the KATP channel pore-forming subunit Kir6.2 (17). Although Kir6.1 protein level was increased by ischemia, this only occurred after prolonged ischemia (60 min of ischemia followed by 24–72 h of reperfusion). Sublethal ischemia (15–30 min followed by 24 h of reperfusion), which is equivalent to the time course of PMA treatment in the present study, did not induce Kir6.1 expression (3). It is therefore less likely that Kir6.1 would contribute to functional mitoKATP channels even though it was found in mitochondria (57). Furthermore, knockout of Kir6.1 gene in intact mice does not disrupt mitoKATP opening (42). Nevertheless, the present study focuses on the regulatory mechanism of Kir6.2-containing KATP channels in mitochondria and does not exclude the possibility that other protein(s) may form the part of KATP channel complex in mitochondria.

The dual distribution at both plasma membrane and mitochondria has been reported for other ion channel proteins, such as Kv1.3 (59), the Ca2+-activated BK potassium channels (55), the voltage-dependent anion channels (9, 19), and connexin43 (10, 39). Interestingly, connexin43 in mitochondria has recently been shown to be increased by IPC, possibly through phosphorylation (10). These channels lack the NH2-terminal mitochondrial targeting sequence but are targeted to mitochondrial inner membrane by unknown mechanisms. Many mitochondrial proteins of the inner membrane such as mitochondrial carriers lack the NH2-terminal targeting sequence but instead contain sorting and targeting information throughout the mature protein, which is hardly recognizable (11, 18). The targeting information seems to comprise several distant amino acids spread throughout the entire protein (63). Similarly, Kir6.2 does not exhibit a classic NH2-terminal mitochondrial targeting presequence according to the prediction from TargetP (21) and may contain unidentified internal targeting sequences. Although specific posttranslational protein modifications and differential splicing have been hypothesized to account for organelle protein targeting, the mechanisms responsible for the simultaneous targeting of a protein to different cellular compartments are not known and are beyond the focus of the present study.

Import of most nucleus-encoded proteins into mitochondria is highly regulated and mediated by mitochondria targeting sequence with the aid of a handful of distinct complexes including molecular chaperones (34). Both heat shock protein (HSP)70 and HSP90, which are linked to cardioprotection, have been shown to facilitate mitochondrial protein import to mitochondria (38, 63–65, 71, 72). HSP90 and HSP70 specifically interact with the mitochondrial protein import receptor TOM70 at the outer membrane and are required for translocation of precursor proteins. Furthermore, PKC has been shown to be involved in HSP-mediated cardioprotection (13, 68). The present finding points to an important mechanism in Kir6.2-containing KATP channel-mediated cardioprotection: PKC{epsilon}-induced mitochondrial import. It should be noted that the present study was not aimed at defining the molecular identity of putative mitoKATP but rather at studying the regulatory mechanism involved in Kir6.2-containing KATP channel trafficking. This observation does not exclude the possibility that a distinct mitoKATP or other KATP component(s) in mitochondria may exist. It has been reported that succinate dehydrogenase may be a part of a mitoKATP macromolecular supercomplex that contributes to cardioprotection (5). A Ca2+-activated K+ channel has also been found in mitochondrial inner membrane and linked to cardioprotection (69).

The evidence supporting a protective role for the Kir6.2-containing KATP channels has been provided by studies using KATP-deficient COS-7 cells and Kir6.2-knockout mice. By cotransfection of Kir6.2/SUR2A genes, Jovanovic et al. (33) demonstrated that delivery of Kir6.2 and SUR2A genes into COS-7 cells resulted in protection against hypoxia-reoxygenation injury as a result of inhibition of intracellular Ca2+ loading. Studies in Kir6.2-knockout mice (28, 58) showed a failure of IPC to reduce infarct size and preserve contractile recovery in Kir6.2-deficient mice. The activation of PKC has also been linked to cardioprotection (50) and KATP channels (40, 66). A number of possibilities have been suggested as to how opening the mitoKATP results in cardioprotection, such as reduced Ca2+ overload in mitochondria by depolarizing {Delta}{Psi}m, mild uncoupling and reduced free radical production, and moderate swelling and increased ATP production (14).

It is known that movement of key proteins into mitochondria during apoptosis is essential for the regulation of apoptosis (12). Mitochondria are increasingly recognized as key players in cell survival (25). Most of the mitochondrial proteins are nuclear encoded, synthesized in the cytosol, and transported to mitochondria by translocases (34, 41, 49). Given that mitochondria are a critical site for cardioprotection, our observations indicate that the dynamic regulation of protein trafficking to mitochondria represents a novel mechanism in cardioprotection of IPC.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by a research grant from the American Heart Association.


   ACKNOWLEDGMENTS
 
We are grateful to S. Seino for providing Kir6.2 and SUR2A clones and to R. Y. Tsien for the kind gifts of Mito-CFP and Mito-YFP constructs. We thank Douglas Pfeiffer for comments on the mitochondrial aspect of the study. We also thank Chen Gu and Ofer Wiser for technical help on the FRET assay and Lily Jan for valuable support and suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Hu, 530 Parks Hall, Div. of Pharmacology, Coll. of Pharmacy, 500 W. 12th Ave., Ohio State Univ., Columbus, OH 43210 (e-mail: hu.175{at}osu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aguilar-Bryan L, Bryan J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 20: 101–135, 1999.[Abstract/Free Full Text]
  2. Aguilar-Bryan L, Clement JP 4th, Gonzalez G, Kunjilwar K, Babenko A, Bryan J. Toward understanding the assembly and structure of KATP channels. Physiol Rev 78: 227–245, 1998.[Abstract/Free Full Text]
  3. Akao M, Otani H, Horie M, Takano M, Kuniyasu A, Nakayama H, Kouchi I, Murakami T, Sasayama S. Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts. J Clin Invest 100: 3053–3059, 1997.[Web of Science][Medline]
  4. Alekseev AE, Hodgson DM, Karger AB, Park S, Zingman LV, Terzic A. ATP-sensitive K+ channel channel/enzyme multimer: metabolic gating in the heart. J Mol Cell Cardiol 38: 895–905, 2005.[CrossRef][Web of Science][Medline]
  5. Ardehali H, Chen Z, Ko Y, Mejia-Alvarez R, Marban E. Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity. Proc Natl Acad Sci USA 101: 11880–11885, 2004.[Abstract/Free Full Text]
  6. Ashcroft FM. Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci 11: 97–118, 1988.[CrossRef][Web of Science][Medline]
  7. Ashcroft SJ, Ashcroft FM. Properties and functions of ATP-sensitive K-channels. Cell Signal 2: 197–214, 1990.[CrossRef][Web of Science][Medline]
  8. Babenko AP, Aguilar-Bryan L, Bryan J. A view of sur/KIR6.X, KATP channels. Annu Rev Physiol 60: 667–687, 1998.[CrossRef][Web of Science][Medline]
  9. Bathori G, Parolini I, Tombola F, Szabo I, Messina A, Oliva M, De Pinto V, Lisanti M, Sargiacomo M, Zoratti M. Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains. J Biol Chem 274: 29607–29612, 1999.[Abstract/Free Full Text]
  10. Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P, Konietzka I, Lopez-Iglesias C, Garcia-Dorado D, Di Lisa F, Heusch G, Schulz R. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res 67: 234–244, 2005.[Abstract/Free Full Text]
  11. Brix J, Rudiger S, Bukau B, Schneider-Mergener J, Pfanner N. Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein. J Biol Chem 274: 16522–16530, 1999.[Abstract/Free Full Text]
  12. Chua BT, Volbracht C, Tan KO, Li R, Yu VC, Li P. Mitochondrial translocation of cofilin is an early step in apoptosis induction. Nat Cell Biol 5: 1083–1089, 2003.[CrossRef][Web of Science][Medline]
  13. Coaxum SD, Martin JL, Mestril R. Overexpression of heat shock proteins differentially modulates protein kinase C expression in rat neonatal cardiomyocytes. Cell Stress Chaperones 8: 297–302, 2003.[CrossRef][Web of Science][Medline]
  14. Costa AD, Quinlan CL, Andrukhiv A, West IC, Jaburek M, Garlid KD. The direct physiological effects of mitoKATP opening on heart mitochondria. Am J Physiol Heart Circ Physiol 290: H406–H415, 2006.[Abstract/Free Full Text]
  15. Cuong DV, Kim N, Joo H, Youm JB, Chung JY, Lee Y, Park WS, Kim E, Park YS, Han J. Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts. Mitochondrion 5: 121–133, 2005.[CrossRef][Web of Science][Medline]
  16. D'Hahan N, Moreau C, Prost AL, Jacquet H, Alekseev AE, Terzic A, Vivaudou M. Pharmacological plasticity of cardiac ATP-sensitive potassium channels toward diazoxide revealed by ADP. Proc Natl Acad Sci USA 96: 12162–12167, 1999.[Abstract/Free Full Text]
  17. Dabrowski M, Tarasov A, Ashcroft FM. Mapping the architecture of the ATP-binding site of the KATP channel subunit Kir6.2. J Physiol 557: 347–354, 2004.[Abstract/Free Full Text]
  18. Davis AJ, Ryan KR, Jensen RE. Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane. Mol Biol Cell 9: 2577–2593, 1998.[Abstract/Free Full Text]
  19. Dermietzel R, Hwang TK, Buettner R, Hofer A, Dotzler E, Kremer M, Deutzmann R, Thinnes FP, Fishman GI, Spray DC, Siemen D. Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes. Proc Natl Acad Sci USA 91: 499–503, 1994.[Abstract/Free Full Text]
  20. Downey JM. The cellular mechanisms of ischemic and pharmacological preconditioning (Abstract). Cardiovasc J S Afr 15: S3, 2004.
  21. Emanuelsson O, Nielsen H, Brunak S, von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016, 2000.[CrossRef][Web of Science][Medline]
  22. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res 81: 1072–1082, 1997.[Abstract/Free Full Text]
  23. Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X, Schindler PA. The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem 271: 8796–8799, 1996.[Abstract/Free Full Text]
  24. Gordon GW, Berry G, Liang XH, Levine B, Herman B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74: 2702–2713, 1998.[Web of Science][Medline]
  25. Green DR, Reed JC. Mitochondria and apoptosis. Science 281: 1309–1312, 1998.[Abstract/Free Full Text]
  26. Gross GJ, Fryer RM. Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning. Circ Res 84: 973–979, 1999.[Abstract/Free Full Text]
  27. Gross GJ, Peart JN. KATP channels and myocardial preconditioning: an update. Am J Physiol Heart Circ Physiol 285: H921–H930, 2003.[Abstract/Free Full Text]
  28. Gumina RJ, Pucar D, Bast P, Hodgson DM, Kurtz CE, Dzeja PP, Miki T, Seino S, Terzic A. Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics. Am J Physiol Heart Circ Physiol 284: H2106–H2113, 2003.[Abstract/Free Full Text]
  29. Hu K, Duan D, Li GR, Nattel S. Protein kinase C activates ATP-sensitive K+ current in human and rabbit ventricular myocytes. Circ Res 78: 492–498, 1996.[Abstract/Free Full Text]
  30. Hu K, Huang CS, Jan YN, Jan LY. ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C. Neuron 38: 417–432, 2003.[CrossRef][Web of Science][Medline]
  31. Hu K, Mochly-Rosen D, Boutjdir M. Evidence for functional role of {epsilon}PKC isozyme in the regulation of cardiac Ca2+ channels. Am J Physiol Heart Circ Physiol 279: H2658–H2664, 2000.[Abstract/Free Full Text]
  32. Jaburek M, Costa AD, Burton JR, Costa CL, Garlid KD. Mitochondrial PKC epsilon and mitochondrial ATP-sensitive K+ channel copurify and coreconstitute to form a functioning signaling module in proteoliposomes. Circ Res 99: 878–883, 2006.[Abstract/Free Full Text]
  33. Jovanovic A, Jovanovic S, Lorenz E, Terzic A. Recombinant cardiac ATP-sensitive K+ channel subunits confer resistance to chemical hypoxia-reoxygenation injury. Circulation 98: 1548–1555, 1998.[Abstract/Free Full Text]
  34. Kaldi K, Neupert W. Protein translocation into mitochondria. Biofactors 8: 221–224, 1998.[Web of Science][Medline]
  35. Kowaltowski AJ, Seetharaman S, Paucek P, Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria. Am J Physiol Heart Circ Physiol 280: H649–H657, 2001.[Abstract/Free Full Text]
  36. Lacza Z, Snipes JA, Miller AW, Szabo C, Grover G, Busija DW. Heart mitochondria contain functional ATP-dependent K+ channels. J Mol Cell Cardiol 35: 1339–1347, 2003.[CrossRef][Web of Science][Medline]
  37. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[CrossRef][Medline]
  38. Latchman DS. Heat shock proteins and cardiac protection. Cardiovasc Res 51: 637–646, 2001.[Abstract/Free Full Text]
  39. Li H, Brodsky S, Kumari S, Valiunas V, Brink P, Kaide J, Nasjletti A, Goligorsky MS. Paradoxical overexpression and translocation of connexin43 in homocysteine-treated endothelial cells. Am J Physiol Heart Circ Physiol 282: H2124–H2133, 2002.[Abstract/Free Full Text]
  40. Light PE, Bladen C, Winkfein RJ, Walsh MP, French RJ. Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels. Proc Natl Acad Sci USA 97: 9058–9063, 2000.[Abstract/Free Full Text]
  41. Lithgow T. Targeting of proteins to mitochondria. FEBS Lett 476: 22–26, 2000.[CrossRef][Web of Science][Medline]
  42. Miki T, Suzuki M, Shibasaki T, Uemura H, Sato T, Yamaguchi K, Koseki H, Iwanaga T, Nakaya H, Seino S. Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1. Nat Med 8: 466–472, 2002.[CrossRef][Web of Science][Medline]
  43. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
  44. Nichols CG, Lederer WJ. Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Am J Physiol Heart Circ Physiol 261: H1675–H1686, 1991.[Abstract/Free Full Text]
  45. Nichols CG, Ripoll C, Lederer WJ. ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction. Circ Res 68: 280–287, 1991.[Abstract/Free Full Text]
  46. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature 305: 147–148, 1983.[CrossRef][Medline]
  47. O'Rourke B. Myocardial KATP channels in preconditioning. Circ Res 87: 845–855, 2000.[Abstract/Free Full Text]
  48. Periasamy A, Diaspro A. Multiphoton microscopy. J Biomed Opt 8: 327–328, 2003.[CrossRef][Web of Science][Medline]
  49. Pfanner N, Meijer M. The Tom and Tim machine. Curr Biol 7: R100–R103, 1997.[CrossRef][Web of Science][Medline]
  50. Ping P, Zhang J, Pierce WM Jr, Bolli R. Functional proteomic analysis of protein kinase C epsilon signaling complexes in the normal heart and during cardioprotection. Circ Res 88: 59–62, 2001.[Abstract/Free Full Text]
  51. Reers M, Smith TW, Chen LB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30: 4480–4486, 1991.[CrossRef][Medline]
  52. Schwanstecher M, Schwanstecher C, Chudziak F, Panten U, Clement JP 4th, Gonzalez G, Aguilar-Bryan L, Bryan J. ATP-sensitive potassium channels. Methods Enzymol 294: 445–458, 1999.[Medline]
  53. Seharaseyon J, Ohler A, Sasaki N, Fraser H, Sato T, Johns DC, O'Rourke B, Marban E. Molecular composition of mitochondrial ATP-sensitive potassium channels probed by viral Kir gene transfer. J Mol Cell Cardiol 32: 1923–1930, 2000.[CrossRef][Web of Science][Medline]
  54. Seino S. ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61: 337–362, 1999.[CrossRef][Web of Science][Medline]
  55. Siemen D, Loupatatzis C, Borecky J, Gulbins E, Lang F. Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line. Biochem Biophys Res Commun 257: 549–554, 1999.[CrossRef][Web of Science][Medline]
  56. Singh H, Hudman D, Lawrence CL, Rainbow RD, Lodwick D, Norman RI. Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes. J Mol Cell Cardiol 35: 445–459, 2003.[CrossRef][Web of Science][Medline]
  57. Suzuki M, Kotake K, Fujikura K, Inagaki N, Suzuki T, Gonoi T, Seino S, Takata K. Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria. Biochem Biophys Res Commun 241: 693–697, 1997.[CrossRef][Web of Science][Medline]
  58. Suzuki M, Li RA, Miki T, Uemura H, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Ogura T, Seino S, Marban E, Nakaya H. Functional roles of cardiac and vascular ATP-sensitive potassium channels clarified by Kir6.2-knockout mice. Circ Res 88: 570–577, 2001.[Abstract/Free Full Text]
  59. Szabo I, Bock J, Jekle A, Soddemann M, Adams C, Lang F, Zoratti M, Gulbins E. A novel potassium channel in lymphocyte mitochondria. J Biol Chem 280: 12790–12798, 2005.[Abstract/Free Full Text]
  60. Tong X, Porter LM, Liu G, Dhar-Chowdhury P, Srivastava S, Pountney DJ, Yoshida H, Artman M, Fishman GI, Yu C, Iyer R, Morley GE, Gutstein DE, Coetzee WA. Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits. Am J Physiol Heart Circ Physiol 291: H543–H551, 2006.[Abstract/Free Full Text]
  61. Towbin H, Gordon J. Immunoblotting and dot immunobinding—current status and outlook. J Immunol Methods 72: 313–340, 1984.[CrossRef][Web of Science][Medline]
  62. Troyan MB, Gilman VR, Gay CV. Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol. Exp Cell Res 233: 274–280, 1997.[CrossRef][Web of Science][Medline]
  63. Truscott KN, Brandner K, Pfanner N. Mechanisms of protein import into mitochondria. Curr Biol 13: R326–R337, 2003.[CrossRef][Web of Science][Medline]
  64. Voos W. A new connection: chaperones meet a mitochondrial receptor. Mol Cell 11: 1–3, 2003.[CrossRef][Web of Science][Medline]
  65. Walker DM, Pasini E, Kucukoglu S, Marber MS, Iliodromitis E, Ferrari R, Yellon DM. Heat stress limits infarct size in the isolated perfused rabbit heart. Cardiovasc Res 27: 962–967, 1993.[Abstract/Free Full Text]
  66. Wang Y, Ashraf M. Role of protein kinase C in mitochondrial KATP channel-mediated protection against Ca2+ overload injury in rat myocardium. Circ Res 84: 1156–1165, 1999.[Abstract/Free Full Text]
  67. Wang Y, Haider HK, Ahmad N, Ashraf M. Mechanisms by which KATP channel openers produce acute and delayed cardioprotection. Vascul Pharmacol 42: 253–264, 2005.[CrossRef][Web of Science][Medline]
  68. Wu JM, Xiao L, Cheng XK, Cui LX, Wu NH, Shen YF. PKC epsilon is a unique regulator for hsp90 beta gene in heat shock response. J Biol Chem 278: 51143–51149, 2003.[Abstract/Free Full Text]
  69. Xu W, Liu Y, Wang S, McDonald T, Van Eyk JE, Sidor A, O'Rourke B. Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science 298: 1029–1033, 2002.[Abstract/Free Full Text]
  70. Yamada M, Isomoto S, Matsumoto S, Kondo C, Shindo T, Horio Y, Kurachi Y. Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+ channel. J Physiol 499: 715–720, 1997.[Abstract/Free Full Text]
  71. Yellon DM, Marber MS. Hsp70 in myocardial ischaemia. Experientia 50: 1075–1084, 1994.[CrossRef][Web of Science][Medline]
  72. Young JC, Hoogenraad NJ, Hartl FU. Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell 112: 41–50, 2003.[CrossRef][Web of Science][Medline]
  73. Zerangue N, Schwappach B, Jan YN, Jan LY. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 22: 537–548, 1999.[CrossRef][Web of Science][Medline]
  74. Zhou M, Tanaka O, Sekiguchi M, He HJ, Yasuoka Y, Itoh H, Kawahara K, Abe H. ATP-sensitive K+-channel subunits on the mitochondria and endoplasmic reticulum of rat cardiomyocytes. J Histochem Cytochem 53: 1491–1500, 2005.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
V. Garg, J. Jiao, and K. Hu
Regulation of ATP-sensitive K+ channels by caveolin-enriched microdomains in cardiac myocytes
Cardiovasc Res, April 1, 2009; 82(1): 51 - 58.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
J. Jiao, V. Garg, B. Yang, T. S. Elton, and K. Hu
Protein Kinase C-{epsilon} Induces Caveolin-Dependent Internalization of Vascular Adenosine 5'-Triphosphate-Sensitive K+ Channels
Hypertension, September 1, 2008; 52(3): 499 - 506.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. D. T. Costa and K. D. Garlid
Intramitochondrial signaling: interactions among mitoKATP, PKC{varepsilon}, ROS, and MPT
Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H874 - H882.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J.-D. Jiao, V. Garg, B. Yang, and K. Hu
Novel functional role of heat shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic preconditioning
Cardiovasc Res, January 1, 2008; 77(1): 126 - 133.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
G.-X. Zhang, X.-M. Lu, S. Kimura, and A. Nishiyama
Role of mitochondria in angiotensin II-induced reactive oxygen species and mitogen-activated protein kinase activation
Cardiovasc Res, November 1, 2007; 76(2): 204 - 212.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/H322    most recent
01035.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garg, V.
Right arrow Articles by Hu, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garg, V.
Right arrow Articles by Hu, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.