AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H1770-H1776, 2006. First published December 16, 2005; doi:10.1152/ajpheart.01084.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/H1770    most recent
01084.2005v1
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 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 ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, M. T.
Right arrow Articles by Bosnjak, Z. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, M. T.
Right arrow Articles by Bosnjak, Z. J.

Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro

Ming Tao Jiang,1 Marko Ljubkovic,1,2 Yuri Nakae,1 Yang Shi,3 Wai-Meng Kwok,1,4 David F. Stowe,1,2 and Zeljko J. Bosnjak1,2

Departments of 1Anesthesiology, 2Physiology, 3Surgery, and 4Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 13 October 2005 ; accepted in final form 15 December 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoKATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoKATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoKATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoKATP channels was usually below 80 pS (~70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoKATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 µM) increased the cumulative open probability of the mitoKATP channel previously inhibited by ATP (P < 0.05), but its inactive analog 4{alpha}-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K+ channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-{delta} in the IMM. These data provide the first characterization of the human cardiac mitoKATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning.

bilayers; mitochondria; protein kinase C; ischemia; preconditioning

THE MITOCHONDRIAL ATP-sensitive K+ (mitoKATP) channel was first discovered in the inner membrane (IMM) of rat liver mitochondria (21). Subsequent studies have confirmed its presence by patch-clamp studies of the mitoplasts (11, 13) or IMM reconstituted in lipid bilayers (1, 5, 27, 28, 31, 45). However, the unitary conductance of the putative mitoKATP channel reported in these studies differed significantly, varying between ~10 and 100 pS. Equally controversial is the proposed molecular composition of the mitoKATP channel (1, 27). Several groups have even questioned the very existence of mitoKATP, based on the measurement of mitochondrial K+ fluxes or volume change (7, 12). In humans, the mitoKATP channel was detected in a T-lymphocyte cell line (11), but the existence and properties of human cardiac mitoKATP channel have not been reported.

Numerous pharmacological studies have shown that the mitoKATP channel plays a critical role in myocardial protection, induced by ischemic (15) and pharmacological preconditioning (14, 39, 44). Ischemic preconditioning (IPC) triggers the translocation of PKC isoforms, such as {delta} or {epsilon}, from the cytosolic to the particulate fraction containing the mitochondria (33, 42, 43). The translocated PKC has been proposed to regulate the mitoKATP channel, and blockade of the PKC transfer prevents IPC in both animals and humans (20, 38, 42, 43). Activation of the mitoKATP channel by phorbol 12-myristate 13-acetate (PMA), a PKC activator, was found to protect the integrity of isolated mitochondria by preventing the opening of permeability transition pore and cytochrome-c release to simulated ischemia (24). Furthermore, there is evidence that PKC activation by PMA potentiates the effect of diazoxide on flavoprotein oxidation, an indirect indicator of mitoKATP channel activity (35). Although these studies suggest a role of PKC in preconditioning, there is no evidence that the mitoKATP channel is directly regulated by cytosolic PKC or other protein kinases due to the physical barrier of outer mitochondrial membrane (OMM). Costa et al. (9) proposed that phosphorylation of a target protein on OMM by protein kinase G (PKG) transmits the cardioprotective signals to PKC-{epsilon} located in the intermembrane space during preconditioning. On the other hand, it is possible that the mitoKATP is regulated by local protein kinase(s) associated with IMM or intermembrane space.

The purpose of our study was to characterize human cardiac mitoKATP channel and to investigate its potential regulation by a local PKC control mechanism. Reconstitution of the human mitoKATP channel in lipid bilayers, away from the cytosolic environment, allowed us to examine whether the mitoKATP channel is directly regulated by PKC located within the IMM.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial isolation. The Institutional Review Board for Clinical Studies at the Medical College of Wisconsin approved this study. The investigation conforms to the principles outlined in the Declaration of Helsinki. Human ventricular muscles were obtained from two nonfailing donor hearts from brain-dead patients. Human atria were obtained during cardiac surgery with informed consent. Cardiac mitochondria were isolated according to the procedure of Solem and Wallace (37), with modifications as described previously (28). Briefly, tissues from the left ventricles or atria were cut into small pieces and homogenized in an ice-cold isolation buffer containing (in mM) 30 MOPS (pH 7.2), 200 mannitol, 50 sucrose, 5 KH2PO4, and 1 EGTA and 0.1% BSA in the presence of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). After centrifugation at 3,000 g for 20 min, the supernatant was centrifuged at 8,000 g to obtain mitochondria (the pellet). Mitochondria were suspended in 3% Percoll in 10% sucrose solution, layered over a 6% Percoll in 20% sucrose solution, and centrifuged at 20,000 g for 30 min. The pellet was resuspended with the isolation medium without EGTA and then stored on ice for preparation of IMM.

Preparation of IMMs. The submitochondrial fraction enriched with IMM was prepared as reported (6, 29). The mitochondrial pellet was osmotically shocked by incubation in 10 mM phosphate buffer (pH 7.4) for 20 min and then in 20% sucrose for another 15 min. Membranes were sonicated (Dual Horn for model 550, Fisher Scientific, Hanover Park, IL) three times for 30 s and centrifuged at 8,000 g for 10 min. The supernatant, containing submitochondrial particles, was fractionated by using a continuous sucrose gradient (30% to 60%) and then centrifuged at 80,000 g overnight. The heavy fraction was resuspended with the isolation medium without EGTA and centrifuged at 380,000 g for 30 min. The final pellet, enriched in IMM, was resuspended in the isolation medium without EGTA and BSA and then stored at –80°C in small aliquots until use.

Reconstitution of mitoKATP channels into lipid bilayers. The vesicles of IMM were reconstituted into lipid bilayers as reported previously (28). Briefly, the IMMs were added to the cis chamber and fused into the lipid bilayers in a symmetrical solution containing (in mM) 30 MOPS (pH 7.4), 150 potassium glutamate, 1 EGTA, 1.03 CaCl2 (free Ca2+, 10 µM), 0.05 K2ATP, and 0.5 MgCl2. Ag/AgCl electrodes were placed into each chamber via agar salt (0.5 M KCl) bridges, and the trans chamber was connected to the head stage of a bilayer clamp amplifier (BC-525C, Warner Instrument, Hamden, CT). The cis chamber was held at virtual ground. Experiments were performed at room temperature. After incorporation of cation channel(s), single channel currents at a holding potential of +30 or +40 mV (trans/cis, –30 or –40 mV by convention) or as indicated were collected by using an Axon Digidata 1332 AD/DA interface (Axon Instruments, Union City, CA) with pClamp software (version 8.01, Axon Instruments). The currents were filtered at 0.5 kHz with an eight-pole Bessel filter and digitized at 2.5 kHz. The channel activity, accumulated over 2 to 4 min, was expressed as cumulative channel open probability (NPo), where N is the apparent number of channels, and Po is the mean open-state probability. NPo was determined from amplitude histograms after multiple Gaussian curve fitting (Origin 6.0, Microcal Software, Northampton, MA), as described previously (28).

Western blot analysis. Western blot analysis assays for sarcolemmal KATP (sarcKATP) channel subunits in mitochondria were conducted as described previously (22) using a 4–20% gradient Criterion Precast Gel (Bio-Rad), and that for PKC isoforms were done by using 7.5% Criterion Precast Gel (Bio-Rad) as described elsewhere (36). The antibodies were obtained from Santa Cruz Biotechnology, including inward rectifying K+ channel (Kir)6.2 (G-16), Kir6.1 (C-16), sulfonylurea receptor (SUR)2 (C-15), SUR1 (C-16), PKC-{epsilon}, PKC-{delta}, and PKC-beta. The secondary antibodies were conjugated with horseradish peroxidase. Detected proteins were visualized by using chemiluminescence. To ensure detection specificity, negative controls were done by using primary antibodies preabsorbed with immunogen peptides in a one-to-five ratio.

Characterization of mitoKATP channel reconstituted in lipid bilayers. MitoKATP channels were identified by their inhibition with ATP and 5-hydroxydecanoic acid (5-HD) and their activation by diazoxide, a mitoKATP channel opener. All modulators were added to the cis chamber and stirred for 30 s.

Effect of PMA on mitoKATP channel opening. After the appearance of K+-conducting current in lipid bilayers, 0.5 mM ATP was used to screen ATP-sensitive channels. PMA (2 µM) was added to the cis chamber while being stirred. The inactive phorbol ester 4{alpha}-phorbol 12,13-didecanoate (PDD) was used as the negative control. Channel activities were monitored for up to 15 min, and the identity of mitoKATP channels was confirmed at the conclusion of the experiment by their inhibition with 5-HD.

Chemicals. The following drugs and chemicals were used: protease inhibitor cocktail, n-decane, MOPS, diazoxide, 5-HD, and ATP (Sigma-Aldrich); BSA (Serologicals, Milwaukee, WI); L-{alpha}-phosphatidylethanolamine and L-{alpha}-phosphatidyl-serine (Avanti Polar-Lipid, Alabaster, AL); and PMA and PDD (Calbiochem). PMA, PDD, and diazoxide were dissolved in DMSO before adding to the experimental solution. The final concentration of DMSO (<0.1%) alone did not exhibit any effect on channel current.

Statistical analysis. Because of significant variance in NPo between groups, data were analyzed after square root transformation and presented as means ± SE. Multiple comparisons between groups were analyzed by analysis of variance, followed by Duncan's range tests. A value of P ≤ 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of human cardiac mitoKATP channel. The human IMM vesicles were fused into the lipid bilayers, and KATP channels were identified based on their inhibition by ATP and/or 5-HD (15, 16, 32), as well as on their activation by diazoxide, a putative agonist selective for the mitoKATP channel (15, 18, 19).

In the presence of 150 mM symmetrical potassium glutamate and at the holding potential of +40 mV, recorded chord conductance of single mitoKATP channels was mostly below 80 pS (n = number of observations): ~20 pS (n = 8), ~40 pS (n = 8), ~60 pS (n = 7), although the channel conductance >80 pS was also recorded (n = 9). Figure 1A shows a K+ channel with a cord conductance of 65 pS at a holding potential of +40 mV. The channel activity was abolished by 5-HD, confirming its identity as a mitoKATP. Figure 1B shows original traces of another mitoKATP recorded at varying holding potentials, and Fig. 1C illustrates its corresponding current-voltage relationship (I-V). Linear regression analysis revealed a slope conductance of 57 pS, similar to that observed for mitoKATP from bovine (45) or rat hearts (28). Figure 2A shows original traces of a mitoKATP channel with a small conductance at various holding potentials. Its I-V curve is presented in Fig. 2B, and calculated slope conductance is 24 pS. We did not observe significant rectification within the range of voltages used, which probably can be better evaluated at higher voltages closer to a physiological range of matrix potential (~180 mV). However, we were limited by the poor stability of artificial bilayers beyond –60 or +60 mV, especially in patches with multiple channels.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. Characteristics of single human cardiac mitochondrial ATP-sensitive K+ (mitoKATP) channel at ~60 pS. A: 60-pS (cord conductance) mitoKATP channel during control [mean open-state probability (Po) = 0.9] and its blockade by 5-hydroxydecanoate (5-HD, 200 µM, Po = 0.03). C, closed state; O, open state. B: voltage-dependent current amplitudes of another mitoKATP channel reconstituted in lipid bilayers. C: current-voltage relationship in symmetrical 150 mM potassium glutamate and 0.5 mM Mg2+. A slope conductance ({gamma}) of 57 pS was obtained from the linear portion of the curve. A slight rectification toward the trans chamber was observed. Data represent 6 observations.

 

Figure 2
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 2. Characteristics of a single human cardiac mitoKATP channel with a slope conductance of 24 pS. A: voltage-dependent current amplitudes of a mitoKATP channel reconstituted in lipid bilayers. B: current-voltage relationship in symmetrical 150 mM potassium glutamate and 0.2 mM Mg2+. A slope conductance of 24 pS was obtained. Data represent 8 observations.

 
The single channel conductance from patches with multiple mitoKATP channels was often better appreciated in the presence of mitoKATP inhibitors ATP or 5-HD. As shown in Fig. 3, at baseline (control, Fig. 3A), the observed peak current was ~6 pA at +30 mV, with a small conductance channel opening at or above 0.75 pA (25 pS). An addition of 0.5 mM ATP (Fig. 3B) inhibited most of the larger flickering channels, but the small channel remained mostly open. Increasing ATP to 1 mM (Fig. 3C) gradually inhibited this channel until complete closure. Fig. 3C, inset, shows the segment of recordings with transition of the small conductance mitoKATP from opening (at two levels, O1 and O2) to closing on an expanded scale. The corresponding amplitude histogram clearly shows a single channel conductance of 25 and 75 pS, with the latter likely representing simultaneous opening of three channels of the 25 pS conductance. These data show that ATP induced both concentration- and time-dependent inhibition of mitoKATP channels, which was frequently observed.


Figure 3
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. ATP-induced steplike inhibition of mitoKATP conductance. At control (A), a cluster of channels was seen open at and above 0.75 pA (25 pS), with peak current of ~6 pA due to the spikes of openings. Subsequent addition of ATP (0.5 mM) into cis (B) inhibited most of the spiking channels, with sustained opening at 0.75 pA. When ATP was raised to 1 mM (C), channels underwent steplike transition to smaller conductance states before complete closure (C); this is clearly shown on an expanded scale (C, left inset) (O2 and O1, open states). Histogram analysis (C, right inset) showed a transition from 75 pS (i.e., O2) to 25 pS (i.e., O1) before closure (C).

 
This transition can also be observed in the presence of another blocker, 5-HD. Figure 4 shows regulation of mitoKATP channels by ATP, GTP, and 5-HD from a different patch of IMM fused in bilayers. At baseline (Fig. 4A), mitoKATP channel openings were active; an addition of 0.5 mM ATP (Fig. 4B) reduced the peak current by ~75% to give a smaller conductance of ~20 pS. An addition of 0.5 mM GTP (Fig. 4C), a mitoKATP channel agonist (32), markedly increased the peak current that exceeded that seen at baseline. 5-HD (200 µM, Fig. 4D) turned the sustained opening into a burst, with steplike conductance levels of ~40, 80, and 120 pS, as shown on an expanded scale (Fig. 4D, inset). This was followed by complete closure (Fig. 4E). Thus similar to ATP, 5-HD also caused a steplike closure of the channels, revealing multiple conductance states. These smaller conductance levels could arise from clusters of smaller channels, or they could represent subconductance states of the larger channels. Similar steplike behavior was also reported for sarcKATP inhibited by glibenclamide (23).


Figure 4
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4. Effects of ATP, GTP, and 5-HD on mitoKATP channels reconstituted in lipid bilayers. Original recordings of mitoKATP channel activities are shown, and upward deflection represents channel opening at +30 mV (trans/cis). A cluster of mitoKATP channels was active at baseline (control, A), inhibited by 0.5 mM ATP (B), and then reactivated by 0.5 mM GTP (C). Addition of 5-HD inhibited channels, which initially appeared in bursts (D), with conductance levels seen at ~40 (O1), 80 (O2), and 120 (O3) pS (D, inset) before complete closure (E). Data represent 5 observations.

 
We also tested the effect of the mitoKATP channel opener diazoxide on some mitoKATP channels with low initial activities (NPo < 0.1). Data from multiple experiments are summarized in Fig. 5. An addition of diazoxide (either 50 or 100 µM) significantly increased NPo from the control levels (P < 0.05), and this current was subsequently blocked by 5-HD (200 µM, P < 0.05 vs. diazoxide). These results confirmed the presence of mitoKATP channels in native human IMM. The effects of diazoxide and 5-HD were similar to those reported in reconstituted bovine or rat cardiac KATP channels (28, 45). These concentrations were chosen because they produced consistent activation (diazoxide) or inhibition (5-HD) in preliminary studies. Similar concentrations were found to be effective for bovine (45) or rat mitoKATP (28) channels.


Figure 5
View larger version (7K):
[in this window]
[in a new window]
  		Fig. 5. Effect of diazoxide and 5-HD on human mitoKATP channels reconstituted in lipid bilayers. Control refers to activities observed under control conditions. Addition of diazoxide (50 or 100 µM) increased cumulative open-state probability (NPo) and subsequent addition of 5-HD (200 µM) blocked diazoxide-induced activation. *P < 0.05 vs. control or 5-HD; n = 6 observations.

 
Because the exact molecular composition of the mitoKATP channel is not known, we explored its potential similarity to the sarcKATP channel. We probed the isolated IMMs with antibodies targeting the subunits of sarcKATP: the SUR (SUR1 and SUR2) and the Kir channel (Kir6.1 and Kir6.2). As shown in Fig. 6 (left), Kir6.2 antibody detected a 56-kDa peptide in human ventricular IMM and in two atrial mitochondrial preparations (Fig. 6, M). The specificity was confirmed by preincubation of the antibody with its antigen peptide (Fig. 6, right). Interestingly, this 56-kDa peptide is similar to the ethanol-extracted 55-kDa mitochondrial protein reported by Miranova et al. (27), which was proposed to be the mitochondrial inward rectifier subunit. However, we were not able to detect Kir6.1, as reported by Lacza et al. (25), or SUR subunits with specificity (not shown).


Figure 6
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6. Detection of inward rectifying K+ channel (Kir)6.2 in mitochondria. Left: 56-kDa peptide was detected by anti-Kir6.2 antibody in human inner mitochondrial membrane (IMM) and atrial mitochondria (M, two separate samples, lanes 2 and 3). Right: same blot was stripped and then incubated with anti-Kir6.2 antibody preabsorbed with antigen peptide. Anti-Kir6.2 antibody failed to redetect the 56-kDa peptide.

 
Regulation of human cardiac mitoKATP by local PKC. To explore whether human mitoKATP channels are regulated by PKC associated with the IMM, we tested the effect of PKC activator PMA on mitoKATP channels reconstituted into the lipid bilayers. Figure 7A shows the original recording of mitoKATP openings at baseline (control), followed by inhibition with 0.5 mM ATP. An addition of PMA (2 µM) in the cis chamber reactivated the channels that were eventually inhibited by 5-HD (400 µM). In contrast to PMA, its inactive analog PDD had no effect (Fig. 7B). Cumulative data from six separate observations are summarized in Fig. 7C. These results confirmed that PMA significantly increases the NPo of mitoKATP channels previously inhibited by ATP (P < 0.05). The effects of PMA on mitoKATP obtained at the single-channel level are in agreement with the effect of PMA on the mitoKATP activity in studies (9, 24) done on isolated mitochondria. Because the mitoKATP channels reconstituted in lipid bilayers are in cytosol-free environment, no cytosolic PKC translocation was thus possible; the effect of PMA was likely mediated by PKC associated with the IMM. To confirm this hypothesis, we screened the IMM for PKC isozymes. As shown in Fig. 7C, inset, we were able to detect PKC-{delta} but not PKC-{epsilon} (or beta, not shown) in human IMM.


Figure 7
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 7. Activation of human mitoKATP channels by phorbol 12-myristate 13-acetate (PMA) in lipid bilayers. A: original recordings of mitoKATP channels in lipid bilayers. ATP at 0.5 mM markedly inhibited channel activities seen at baseline (control); addition of PMA (2 µM) reactivated the channels after ~6 min, which were then completely blocked by 5-HD (200 or 400 µM). B: inactive analog of PMA, 4{alpha}-phorbol 12,13-didecanoate (PDD, 2 µM), failed to activate mitoKATP channels inhibited by ATP (0.5 mM), but subsequent addition of PMA reactivated the mitoKATP activity (representative of 3 observations). C: summarized data on mitoKATP channel activation by PMA. This PKC activator significantly increased mitoKATP NPo that was previously inhibited by ATP. *P < 0.05 vs. control or PMA; n = 6 observations. C, inset: Western blot analysis showing detection of ~80-kDa PKC-{delta}, but not PKC-{epsilon}, in human IMM. Data represent 2 determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To our knowledge, this is the first study characterizing the human cardiac mitoKATP channel and its modulation by PMA. Our results demonstrate that the mitoKATP channels are present in human cardiac mitochondria, activated by diazoxide, and inhibited by ATP and 5-HD. Moreover, the mitoKATP channels reconstituted in lipid bilayers can be directly stimulated by the PKC activator PMA without translocation of cytosolic kinases. Western blot analysis demonstrated that Kir6.2 immunoreactive protein and PKC-{delta} are present in human IMM. These data suggest that the mitoKATP channel is regulated locally by PKC associated with the IMM. This local control mechanism may serve as an alternative signal transduction pathway for the mitoKATP channel activation during preconditioning to that proposed for PKC translocated from the cytosol to the mitochondria (33, 34, 41, 42).

The existence of the mitoKATP channel was first reported by Inoue et al. (21) in a patch-clamp study on liver mitoplasts. In the present study, we observed multiple conductances of the mitoKATP channels (mostly below 80 pS), similar to previous observations in the rat heart (28). The conductance levels of these channels, however, are modulated by 5-HD as well as ATP. In mitochondria isolated from human lymphocyte cell line, Dahlem et al. (11) observed an outwardly rectifying K+ channel with a slope conductance of 82 pS at positive test potentials and 15 pS at negative test potentials. This channel, however, responded poorly to ATP inhibition: it was only partially inhibited by 12.5 mM ATP, whereas a higher ATP concentration had no effect. In our experiments, 0.5 mM or 1 mM ATP was effective in blocking most of the mitoKATP channels.

A variety of mitoKATP channel conductances have been reported since its initial discovery. In mitochondria from bovine hearts reconstituted in lipid bilayers, recorded mitoKATP conductance was 30 pS (31) and 56 pS (45). In mitoplasts prepared from rat cardiomyocytes, Er et al. (13) observed a 13-pS channel that was voltage dependent with no rectification. Ardehali et al. (1) observed a mitoKATP channel at 200 pS in 500 mM K+ and <100 pS in 100 mM K+. Szewczyk and coworkers (4, 5) recorded the mitoKATP channel with 103-pS conductance that was reduced by over 50% in the presence of 1 mM Mg2+. Different observations may result from differences in preparations, concentrations of charge carriers, as well as the presence of modulators such as Mg2+ and ATP. They also reflect the complexity of studying the mitoKATP channels and raise the possibility that there may be more than one type of channel that respond to known activators and inhibitors of mitoKATP channels. Although we detected mitoKATP channels at ~20 pS, channels with smaller conductance at ~10 pS could not be resolved under holding voltages used in our observations. Nevertheless, the results from our study revealed multiple conductance states of the human mitoKATP channel. Whether they represent clusters of mitoKATP channels or multiple-conducting states of a single channel has yet to be determined.

MitoKATP channel has not yet been identified at the molecular level. Several studies (10, 25) that attempted to reveal its molecular structure relied on probing the mitochondria with antibodies against sarcKATP subunits. However, this approach is often criticized due to lack of proper negative controls with antigen peptide competition, which may show false positive findings (7). In the present study, anti-Kir6.2 antibody detected a 56-kDa protein in human cardiac IMM, but we failed to detect Kir6.1 and SUR subunits in the same sample with specificity. Garlid and coworkers (3, 27) have extracted the putative mitochondrial KIR and mitochondrial SUR from mitochondria and proposed a molecular structure similar to sarcKATP (3, 27). An alternative molecular complex, which consists of five mitochondrial proteins within the IMM [the ATP binding cassette protein, a phosphate carrier, adenosine nucleotide translocator (ANT), ATP synthase, and succinate dehydrogenase], has been recently proposed (1). Thus the definition of the mitoKATP channel molecular nature remains far from resolved. Moreover, the very existence of the mitoKATP channel has been challenged, based on measurements of mitochondrial volume change, K+ fluxes, and respiration in the presence of diazoxide or 5-HD (7, 12).

Activation of the mitoKATP channel by PKC has been considered a crucial event in cardiac preconditioning (17, 30). Although it has been reported that PKC-{epsilon} translocates to the particulate fraction during IPC (33, 42, 43), there is no evidence that cytosolic PKC isozymes can permeate the OMM and interact directly with the mitoKATP in the IMM. The role of mitochondrial PKC in the mitoKATP channel regulation was suggested in the study (24) on isolated mitochondria, which are devoid of cytosolic components. The authors of that study observed that PKC activation by PMA mimics the effect of diazoxide in preventing permeability pore transition, which was abolished by the mitoKATP inhibitor 5-HD. Costa et al. (9) proposed that PKG may phosphorylate some target protein on the OMM and transmit the cardioprotective signals from cytosol to IMM via PKC-{epsilon} located in the intermembrane space. The evidence for this proposal, however, was based only on pharmacological studies in isolated mitochondria, using changes in mitochondrial volume and respiration as indirect indexes of the mitoKATP channel activity.

In our experiments, PMA-activated mitoKATP channels reconstituted in lipid bilayers, and we were able to detect PKC-{delta} in IMM. These data suggest that PKC is associated with the IMM where it can directly activate mitoKATP channels, probably by forming a signaling module. Further study is needed to investigate whether Kir6.2 is physically bound with PKC-{delta} in IMM. PKC was previously shown to phosphorylate the Kir subunits in sarcKATP (26), and a similar mechanism may apply to mitoKATP. This phosphorylation likely reduced the sensitivity of mitoKATP to ATP inhibition, as well as 5-HD inhibition (Fig. 7).

In the mitochondria, the role of PKC is not limited to regulating the mitoKATP channels. PKC-{delta} was also shown recently to bind specifically with pyruvate dehydrogenase and to prevent its reactivation during reperfusion (8), although the source (cytosolic vs. mitochondrial) of the PKC remains elusive. PKC-{epsilon}, although not detected in the present study with human IMM, has been shown to form a functional module with components of the permeability transition pore from mouse mitochondria, including the voltage-dependent anion channel (VDAC) located on the OMM and the ANT located on the IMM (2, 40). The VDAC, located on the OMM, is obviously a better target for cytosolic kinases, such as PKCs, than the mitoKATP located in the IMM. On the other hand, ANT may also form a part of the proposed mitoKATP complex (1); its interaction with PKC-{epsilon} may also regulate mitoKATP activity.

PKC-{epsilon} was also shown recently to interact with cytochrome-c oxidase (19) and with a scaffold protein cypher and ANT on the IMM (40). This evidence suggests that PKC (and probably other kinases), located within the cardiac mitochondria, may play an important role in regulating respiration as well as ionic homeostasis. In the intact cell, a soluble second messenger, such as the diacylglycerol or reactive oxygen species, may activate mitochondrial PKC during IPC.

In summary, we have provided the first characterization of the human cardiac mitoKATP channel and its regulation by PMA in vitro. Our data suggest that the mitoKATP channel is activated by PKC associated with the IMM. This local control mechanism may represent an alternative regulatory pathway to that proposed for cytosolic PKC translocated to mitochondria during preconditioning. Further studies are needed to validate this local control mechanism.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by American Heart Association Northland Affiliate (006043Z to M. T. Jiang and 355608Z to D. F. Stowe), National Institutes of Health (GM-066730 and HL-034708 to Z. J. Bosnjak and HL-58691–01 to D. F. Stowe), the Department of Anesthesiology, Medical College of Wisconsin, and Sapporo Medical University, Sapporo, Japan (postdoctoral fellowship support to Y. Nakae).


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Hector H. Valdivia of the University of Wisconsin-Madison who kindly provided some heart tissues used in the current study. We thank Betty Liu and Xiangping Fu for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. Jiang, Dept. of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: mtjiang{at}mcw.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
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ardehali H, Chen Z, Ko Y, Mejia-Alvarez R, and 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]
  2. Baines CP, Song CX, Zheng YT, Wang GW, Zhang J, Wang OL, Guo Y, Bolli R, Cardwell EM, and Ping P. Protein kinase C{epsilon} interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ Res 92: 873–880, 2003.[Abstract/Free Full Text]
  3. Bajgar R, Seetharaman S, Kowaltowski AJ, Garlid KD, and Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain. J Biol Chem 276: 33369–33374, 2001.[Abstract/Free Full Text]
  4. Bednarczyk P, Dolowy K, and Szewczyk A. Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart. FEBS Lett 579: 1625–1632, 2005.[CrossRef][ISI][Medline]
  5. Bednarczyk P, Kicinska A, Kominkova V, Ondrias K, Dolowy K, and Szewczyk A. Quinine inhibits mitochondrial ATP-regulated potassium channel from bovine heart. J Membr Biol 199: 63–72, 2004.[CrossRef][ISI][Medline]
  6. Beutner G, Sharma VK, Giovannucci DR, Yule DI, and Sheu SS. Identification of a ryanodine receptor in rat heart mitochondria. J Biol Chem 276: 21482–21488, 2001.[Abstract/Free Full Text]
  7. Brustovetsky T, Shalbuyeva N, and Brustovetsky N. Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain non-synaptosomal mitochondria. J Physiol (Lond) 568: 47–59, 2005.[Abstract/Free Full Text]
  8. Churchill EN, Murriel CL, Chen CH, Mochly-Rosen D, and Szweda LI. Reperfusion-induced translocation of {delta}PKC to cardiac mitochondria prevents pyruvate dehydrogenase reactivation. Circ Res 97: 78–85, 2005.[Abstract/Free Full Text]
  9. Costa ADT, Garlid KD, West IC, Lincoln TM, Downey JM, Cohen MV, and Critz SD. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ Res 97: 329–336, 2005.[Abstract/Free Full Text]
  10. Cuong DV, Kim N, Joo H, Youm JB, Chung JY, Lee Y, Park WS, Kim E, Park YS, and Han J. Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts. Mitochondrion 5: 121–133, 2005.[CrossRef][ISI][Medline]
  11. Dahlem YA, Horn TF, Buntinas L, Gonoi T, Wolf G, and Siemen D. The human mitochondrial KATP channel is modulated by calcium and nitric oxide: a patch-clamp approach. Biochim Biophys Acta 1656: 46–56, 2004.[Medline]
  12. Das M, Parker JE, and Halestrap AP. Matrix volume measurements challenge the existence of diazoxide/glibencamide-sensitive KATP channels in rat mitochondria. J Physiol (Lond) 547: 893–902, 2003.[Abstract/Free Full Text]
  13. Er F, Michels G, Gassanov N, Rivero F, and Hoppe UC. Testosterone induces cytoprotection by activating ATP-sensitive K+ channels in the cardiac mitochondrial inner membrane. Circulation 110: 3100–3107, 2004.[Abstract/Free Full Text]
  14. Fryer RM, Hsu AK, Eells JT, Nagase H, and Gross GJ. Opioid-induced second window of cardioprotection: potential role of mitochondrial KATP channels. Circ Res 84: 846–851, 1999.[Abstract/Free Full Text]
  15. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, and 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]
  16. Garlid KD, Paucek P, Yarov-Yarovoy V, Sun X, and Schindler PA. The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem 271: 8796–8799, 1996.[Abstract/Free Full Text]
  17. Gross GJ and Fryer RM. Mitochondrial KATP channels: triggers or distal effectors of ischemic or pharmacological preconditioning? Circ Res 87: 431–433, 2000.[Free Full Text]
  18. Grover GJ and Atwal KS. Pharmacologic profile of the selective mitochondrial-KATP opener BMS-191095 for treatment of acute myocardial ischemia. Cardiovasc Drug Rev 20: 121–136, 2002.[ISI][Medline]
  19. Grover GJ, D'Alonzo AJ, Darbenzio RB, Parham CS, Hess TA, and Bathala MS. In vivo characterization of the mitochondrial selective KATP opener (3R)-trans-4-[(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)dimethyl-2H-1-benzopyran-6-carbonitril monohydrochloride] (BMS-191095): cardioprotective, hemodynamic, and electrophysiological effects. J Pharmacol Exp Ther 303: 132–140, 2002.[Abstract/Free Full Text]
  20. Hassouna A, Matata BM, and Galinanes M. PKC-{epsilon} is upstream and PKC-{alpha} is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium. Am J Physiol Cell Physiol 287: C1418–C1425, 2004.[Abstract/Free Full Text]
  21. Inoue I, Nagase H, Kishi K, and Higuti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature 352: 244–247, 1991.[CrossRef][Medline]
  22. Jiang MT, Lokuta AJ, Farrell EF, Wolff MR, Haworth RA, and Valdivia HH. Abnormal Ca2+ Release, but normal ryanodine receptors, in canine and human heart failure. Circ Res 91: 1015–1022, 2002.[Abstract/Free Full Text]
  23. Ju YK and Saint DA. Subconductance states of the cardiac KATP channel revealed by partial block with glybenclamide. Pflügers Arch 442: 867–873, 2001.[CrossRef][ISI][Medline]
  24. Korge P, Honda HM, and Weiss JN. Protection of cardiac mitochondria by diazoxide and protein kinase C: implications for ischemic preconditioning. Proc Natl Acad Sci USA 99: 3312–3317, 2002.[Abstract/Free Full Text]
  25. Lacza Z, Snipes JA, Miller AW, Szabo C, Grover AJ, and Busija DW. Heart mitochondria contain functional ATP-dependent K+ channels. J Mol Cell Cardiol 35: 1339–1345, 2002.
  26. Light PE, Bladen C, Winkfein RJ, Walsh MP, and 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]
  27. Mironova GD, Negoda AE, Marinov BS, Paucek P, Costa ADT, Grigoriev SM, Skarga YY, and Garlid KD. Functional distinctions between the mitochondrial ATP-dependent K+ channel (mitoKATP) and its inward rectifier subunit (mitoKIR). J Biol Chem 279: 32562–32568, 2004.[Abstract/Free Full Text]
  28. Nakae Y, Kwok WM, Bosnjak ZJ, and Jiang MT. Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers. Am J Physiol Heart Circ Physiol 284: H1865–H1871, 2003.[Abstract/Free Full Text]
  29. Ohlendieck K, Riesinger I, Adams V, Krause J, and Brdiczka D. Enrichment and biochemical characterization of boundary membrane contact sites from rat-liver mitochondria. Biochim Biophys Acta 860: 672–689, 1986.[Medline]
  30. O'Rourke B. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circ Res 94: 420–432, 2004.[Abstract/Free Full Text]
  31. Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, and Garlid KD. Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria. J Biol Chem 267: 26062–26069, 1992.[Abstract/Free Full Text]
  32. Paucek P, Yarov-Yarovoy V, Sun X, and Garlid KD. Inhibition of the mitochondrial KATP channel by long-chain acyl-CoA esters and activation by guanine nucleotides. J Biol Chem 271: 32084–32088, 1996.[Abstract/Free Full Text]
  33. Ping P, Zhang J, Qiu Y, Tang XL, Manchikalapudi S, Cao X, and Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res 81: 404–414, 1997.[Abstract/Free Full Text]
  34. Qiu Y, Ping P, Tang XL, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu WJ, Teschner S, and Bolli R. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved. J Clin Invest 101: 2182–2198, 1998.[ISI][Medline]
  35. Sato T, O'Rourke B, and Marban E. Modulation of mitochondrial ATP-dependent K+ channels by protein kinase C. Circ Res 83: 110–114, 1998.[Abstract/Free Full Text]
  36. Shi Y, Pritchard KA Jr, Holman P, Rafiee P, Griffith OW, Kalyanaraman B, and Baker JE. Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3. Free Radic Biol Med 29: 695–703, 2000.[CrossRef][ISI][Medline]
  37. Solem LE and Wallace KB. Selective activation of the sodium-independent, cyclosporin A-sensitive calcium pore of cardiac mitochondria by doxorubicin. Toxicol Appl Pharmacol 121: 50–57, 1993.[CrossRef][ISI][Medline]
  38. Speechly-Dick ME, Grover GJ, and Yellon DM. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res 77: 1030–1035, 1995.[Abstract/Free Full Text]
  39. Tanaka K, Ludwig LM, Kersten JR, Pagel PS, and Warltier DC. Mechanisms of cardioprotection by volatile anesthetics. Anesthesiology 100: 707–721, 2004.[CrossRef][ISI][Medline]
  40. Vondriska TMZJ, Wang G, Lu M, Zong N, Pantaleon D, Chen J, and Ping P. Cardioprotective scaffold protein cypher: role to mediate mitochondrial protein interactions (Abstract). Circulation 110: III-205, 2004.
  41. Wang Y and 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]
  42. Wang Y, Hirai K, and Ashraf M. Activation of mitochondrial ATP-sensitive K+ channel for cardiac protection against ischemic injury is dependent on protein kinase C activity. Circ Res 85: 731–741, 1999.[Abstract/Free Full Text]
  43. Wang Y, Takashi E, Xu M, Ayub A, and Ashraf M. Downregulation of protein kinase C inhibits activation of mitochondrial KATP channels by diazoxide. Circulation 104: 85–90, 2001.[Abstract/Free Full Text]
  44. Zaugg M, Lucchinetti E, Spahn DR, Pasch T, and Schaub MC. Volatile anesthetics mimic cardiac preconditioning by priming the activation of mitochondrial KATP channels via multiple signaling pathways. Anesthesiology 97: 4–14, 2002.[CrossRef][ISI][Medline]
  45. Zhang DX, Chen YF, Campbell WB, Zou AP, Gross GJ, and Li PL. Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ Res 89: 1177–1183, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


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
 Anesth. Analg.Home page
M. T. Jiang, Y. Nakae, M. Ljubkovic, W.-M. Kwok, D. F. Stowe, and Z. J. Bosnjak
Isoflurane Activates Human Cardiac Mitochondrial Adenosine Triphosphate-Sensitive K+ Channels Reconstituted in Lipid Bilayers
Anesth. Analg., October 1, 2007; 105(4): 926 - 932.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Comelli, G. Metelli, and I. Mavelli
Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug
Am J Physiol Heart Circ Physiol, February 1, 2007; 292(2): H820 - H829.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
J. D. McCully, Y. Toyoda, H. Wakiyama, A. J. Rousou, R. A. Parker, and S. Levitsky
Age- and gender-related differences in ischemia/reperfusion injury and cardioprotection: effects of diazoxide.
Ann. Thorac. Surg., July 1, 2006; 82(1): 117 - 123.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. J. Grover
Mitochondrial ATP-sensitive potassium channels and mitochondrial protein kinase C: sometimes it's good to have a close neighbor
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1752 - H1753.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/H1770    most recent
01084.2005v1
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 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 ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, M. T.
Right arrow Articles by Bosnjak, Z. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, M. T.
Right arrow Articles by Bosnjak, Z. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.