Published online before print November 8, 2001, doi: 10.1161/hh2401.101752
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© 2001 American Heart Association, Inc.
Cellular Biology |
Characteristics and Superoxide-Induced Activation of Reconstituted Myocardial Mitochondrial ATP-Sensitive Potassium Channels
From the Department of Pharmacology and Toxicology (D.X.Z., Y.-F.C., W.B.C., G.J.G., P.-L.L.) and the Department of Physiology (A.-P.Z.), Medical College of Wisconsin, Milwaukee, Wis.
Correspondence to Pin-Lan Li, MD, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail pli{at}mcw.edu
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Abstract |
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Mitochondrial ATP-sensitive potassium (mitoKATP) channels have been suggested as triggers and end effectors in myocardial ischemic preconditioning. However, the intracellular mechanism regulating mitoKATP channels remains unclear. In the present study, mitoKATP channels from bovine ventricular myocardium were reconstituted using planar lipid bilayers, and the effect of superoxide (O2-·) on the activity of these reconstituted channels was examined. After incorporation, a potassium-selective current was recorded. The mean conductance of this current was 56 pS at 150 mmol/L KCl, which was substantially inhibited by 1 mmol/L MgATP. 5-Hydroxydecanoate (5-HD, 10 to 100 µmol/L), a selective mitoKATP antagonist, reduced the open state probability (NPo) of these channels in a concentration-dependent manner, whereas diazoxide (10 µmol/L), a selective mitoKATP agonist, significantly increased channel activity. HMR-1098 (100 µmol/L), a selective sarcolemmal KATP antagonist, had no effect on the activity of reconstituted channels. Addition of xanthine/xanthine oxidase (100 µmol/L per 0.038 U/mL), an O2-·-generating system, resulted in a marked activation of mitoKATP channels; the NPo of the channels was increased from 0.60±0.10 to 1.94±0.02. This O2-·-induced channel activation was completely abolished by pretreatment with 5-HD (100 µmol/L) or a sulfhydryl alkylating compound, N-ethylmaleimide (2 mmol/L). It is concluded that myocardial mitoKATP channels can be reconstituted into lipid bilayers and that O2-· activates these channels. The effect of O2-· may be associated with its direct action on the sulfhydryl groups of the channel protein.
Key Words: ATP-sensitive K+ channel • mitochondria • superoxide • heart • channel reconstitution
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Introduction |
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Single or multiple brief periods of myocardial ischemia confer protection against infarction produced by a subsequent prolonged ischemia, a phenomenon termed ischemic preconditioning (IPC).1 Although a variety of intracellular signaling pathways and molecules have been implicated in the protective effect of IPC, there is substantial evidence suggesting that the adenosine 5'-triphosphate–sensitive potassium (KATP) channel is an important component of this phenomenon and may serve as a distal effector in this process.2 Cardiac myocytes contain 2 distinct forms of KATP channels, as follows: the sarcolemmal KATP (sarcKATP) channel3 and the mitochondrial ATP-sensitive potassium (mitoKATP) channel.4 The mitoKATP channels are present in the mitochondrial inner membrane and modulate mitochondrial function by regulating mitochondrial membrane potential, ion homeostasis, and matrix volume.5 Recent studies suggest that mitoKATP channels more importantly contribute to IPC-induced cardioprotection compared with sarcKATP channels.6,7 It has been demonstrated that diazoxide, a selective mitoKATP agonist,8 mimicked the beneficial effect of IPC to reduce infarct size or cell killing of cardiomyocytes and that sodium 5-hydroxydecanoate (5-HD), a selective mitoKATP antagonist,9 blocked the protection produced by IPC and diazoxide.10–12 In contrast, selective sarcKATP channel agonists or antagonists were without effect on IPC.13,14
Previous work has shown that IPC-induced protection is associated with an early increase in reactive oxygen species (ROS) during the preconditioning period in hypoxic cardiomyocytes.15,16 Exogenous oxidants have been found to induce preconditioning in the intact heart,17 whereas antioxidants can block the protective effect of IPC.18–20 These results suggest that ROS may be involved in the activation of IPC. However, it remains unknown whether the role of ROS in IPC is attributed to the activation of mitoKATP channels. Given that the mitoKATP channel may serve as a trigger and distal effector for various intracellular signaling pathways in IPC, we hypothesize that ROS may participate in IPC through activation of mitoKATP channels. The present study was designed to characterize myocardial mitoKATP channels using a lipid bilayer reconstitution technique and to determine the effect of ROS on the activity of these reconstituted channels.
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Materials and Methods |
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Preparation of Submitochondrial Membrane Vesicles
Mitochondria were isolated from bovine hearts by differential centrifugation as reported by Holmuhamedov et al.5 Briefly, a piece of left ventricular wall was rapidly dissected from fresh bovine hearts obtained from a local abattoir and immersed in ice-cold isolation buffer containing the following (in mmol/L): sucrose 50, mannitol 200, KH2PO4 5, EGTA 1, and MOPS 5, and 0.1% BSA (pH 7.15 adjusted with KOH). This myocardial section was transported immediately to the laboratory, weighed, and minced into small pieces. The minced tissue was rinsed clear of blood with cold isolation buffer and transferred to a glass Potter-Elvehjem homogenizing vessel on ice. Three 20-second homogenization cycles were performed on ice, and then the tissue suspension was centrifuged at 750g for 10 minutes to remove cellular debris and nuclei. The supernatant containing the mitochondrial fraction was further centrifuged at 8000g for 10 minutes, and the pellet was washed twice by resuspension in isolation buffer followed by centrifugation at 8000g for 10 minutes. To further purify the mitochondrial preparation, an aliquot of mitochondrial suspension (0.5 mL) was layered on top of 15 mL of a solution consisting of 30% Percoll, 0.25 mol/L sucrose, 1 mmol/L EDTA, and 10 mmol/L HEPES (pH 7.4), followed by centrifugation at 35 000g for 30 minutes.21 The major mitochondrial layer was separated from less-dense contaminants and broken mitochondria, collected with a pipette, and washed twice in 5 mL of the isolation buffer by centrifugation at 8000g for 5 minutes. The final mitochondrial pellet was resuspended at 10 to 20 mg protein/mL in the isolation buffer (without EGTA). To prepare the submitochondrial membrane vesicles, the above mitochondrial suspension was sonicated at 55 W for eight 15-second intervals on ice.22 The membrane preparations were aliquoted, frozen in liquid N2, and stored at -80°C until used. All procedures were performed at 4°C, and protein concentrations were determined using the Bio-Rad protein assay with BSA as a standard.
Reconstitution of mitoKATP Channels Into Lipid Bilayers
The mitoKATP channels in mitochondrial membrane vesicles were reconstituted into a planar lipid bilayer as described by Yarov-Yarovoy et al23 and our laboratory.24 A lipid solution of phosphatidylethanolamine and phosphatidylserine (1:1 dissolved in n-decane to 20 mg/mL) was used for channel reconstitution. Briefly, lipid bilayers were painted via a glass rod across an aperture (250 µm in diameter) in the wall of a 1.5-mL Delrin cup that was inserted into a cutaway polyvinyl chloride (PVC) block. The cup formed the trans compartment, and the remainder of the PVC block formed the cis compartment. The cis and trans compartments were initially filled with 50 mmol/L KCl in 20 mmol/L Tris-HCl buffer, pH 7.2. After bilayer formation, an asymmetric KCl gradient (150 mmol/L KCl cis and 50 mmol/L trans) was established by replacing an aliquot of 50 mmol/L KCl with 1.25 mol/L KCl, and the submitochondrial membrane vesicles (50 to 100 µg protein) were added into the cis side. This KCl gradient was used to facilitate the fusion of channel protein into the lipid bilayer. Fusion was induced by application of 40 to 50 mV across the membrane without CaCl2 in solutions. When channel currents were detected, KCl in the trans side was quickly raised to 150 mmol/L to collapse the chemical gradient and prevent further fusion of vesicles into the lipid bilayer.
Recording of mitoKATP Currents
An integrating bilayer clamping amplifier (model BC-525C, Warner Instrument Corp) was used to record single-channel currents. The cis compartment was the voltage control side connected by Ag/AgCl electrode in agar salt bridges to the head stage of the amplifier, while the trans side was held at virtual ground. The amplifier output signals were filtered at 1 kHz using an eight-pole Bessel filter (Frequency Devices). Currents were digitized at a sampling rate of 10 kHz and acquired and stored with an IBM PC equipped with a DigiData 1200 AD/DA interface and pClamp 7.0 software (Axon Instruments). Channel open probability (NPo) was determined from recordings of 3 to 5 minutes as described previously in our bilayer clamping studies.24 A positive current reflects the flow of cations from the cis to trans compartment or the flow of anions in the opposite direction. The holding potential (Vm) is defined as the electric potential of the cis with reference to the trans (ground) compartment.
To establish current-voltage relations of reconstituted channels, the lipid bilayer was exposed to a symmetrical (150/150 mmol/L KCl for cis/trans) or asymmetrical potassium (150/50 mmol/L KCl for cis/trans) solution. N-methyl-D-glucamine was used to compensate for the osmolarity changes of the trans solution. The single-channel currents were then recorded while holding potentials were varied from -40 to +40 mV in steps of 20 mV. Na2ATP with MgCl2 (MgATP, 0.1 to 1 mmol/L); GTP (0.1 to 1 mmol/L); and, in µmol/L, 5-HD (10 to 100), glibenclamide (10 to 100), and diazoxide (10) were used as mitoKATP activators or inhibitors to characterize reconstituted channels in the lipid bilayer. HMR-1098, a selective sarcKATP antagonist,14 was used to distinguish mitoKATP from sarcKATP channels. To determine the effect of O2-· on the activity of mitoKATP channels, xanthine (100 µmol/L)/xanthine oxidase (0.038 U/mL), a commonly used O2-·-generating system, was used in the absence or presence of 5-HD (100 µmol/L), glibenclamide (100 µmol/L), MgATP (1 mmol/L), or the sulfhydryl alkylating compound N-ethylmaleimide (2 mmol/L). Unless otherwise stated, all compounds used were added to the cis solution, and the bilayer potential was held at -40 mV. All experiments were performed at room temperature (
20°C).
Statistics
Data are presented as mean±SEM; the significance of the differences in mean values between and within multiple groups was examined using ANOVA for repeated measures followed by a Duncan multiple-range test. The Student t test was used to evaluate statistical significance of differences between two paired observations. P<0.05 was considered statistically significant.
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Activity of Reconstituted Myocardial mitoKATP Channels in Lipid Bilayer
Figure 1A shows representative recordings of reconstituted K+ currents in lipid bilayers under symmetrical K+ (150/150 mmol/L for cis/trans) at a holding potential of -40 mV. Under these conditions, a negative voltage was applied to the cis side and K+ moved from trans solution to cis solution, thereby forming an outward current. This outward current indicates the movement of K+ from the inside of mitochondria into the cytosol. As shown in Figure 1B, the majority of these K+ currents (
85%) had amplitudes of 1.7 to 3.0 pA. Some smaller channel currents (0.7 to 1.6 pA, 10%) and bigger channel currents (3.1 to 4.0 pA, 5%) were also observed. By determining the voltage dependence of these K+ currents, it was found that the majority of these currents exhibited rectifying properties (Figure 1C). The amplitude of these currents increased linearly with voltage in the negative range, with a mean slope conductance of 56 pS. In some experiments, small conductance channels (ie, 18 pS) and big conductance channels (ie, 100 pS) were observed at 150 mmol/L KCl. Because these currents were rare in our bilayer preparation, we did not carefully characterize them. The following experiments were focused on the 56-pS channels. In the presence of asymmetrical K+ (150/50 mmol/L for cis/trans), the currents exhibited cation selectivity as evidenced by positive currents when the holding potential was at 0 mV. The reversal potential, estimated by linear fitting to the zero current, was -23 mV in the presence of 150/50 mmol/L K+ (Figure 1D). Given the equilibrium potential for K+ (EK) of -29 mV, this indicates that this channel obtained from the mitochondrial membrane is primarily permeable to K+. The NPo of these 56-pS K+ channels was usually <0.8. Occasionally, the channels were fully open with an NPo >0.95.
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Pharmacological Characteristics of Reconstituted Myocardial mitoKATP Channels
Several KATP channel agonists or antagonists were used to further characterize and identify the K+ currents in lipid bilayers as mitoKATP channels. First, the sensitivity of K+ currents to ATP and GTP was examined. Figure 2A depicts representative recordings of mitochondrial K+ currents before and after the sequential addition of MgATP and GTP into the trans solution. MgATP at 1 mmol/L resulted in a marked inactivation of the channels within 2 minutes, and the subsequent addition of 1 mmol/L GTP reactivated the channels. MgATP had no significant effect on the channel activity when added to the cis solution. Figure 2B summarizes the results of these experiments. The NPo was decreased from 0.76±0.08 in control to 0.20±0.06 in the presence of 1 mmol/L ATP, and then restored to 0.77±0.06 by 1 mmol/L GTP. Lower concentrations of ATP (0.5 mmol/L) or GTP (0.1 to 0.5 mmol/L) were also found to decrease or increase channel activity, respectively (data not shown). Figure 2C shows that ATP (1 mmol/L) inhibited the 1.7- to 3.0-pA currents but not the smaller currents. GTP (1 mmol/L) restored the 1.7- to 3.0-pA currents.
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The effects of 5-HD and diazoxide on channel activity were examined. As shown in Figures 3A and 3B, 5-HD (100 µmol/L) added to the cis solution resulted in inactivation of the channels within 3 minutes, and channels could be reactivated by diazoxide (10 µmol/L). 5-HD (10 to 100 µmol/L) concentration-dependently decreased the NPo of the reconstituted channels from 0.66±0.11 to 0.09±0.05, and the NPo was restored to 0.71±0.11 by addition of 10 µmol/L diazoxide (Figure 3B). Diazoxide also significantly activated the channels in the absence of 5-HD (data not shown). Like ATP, 5-HD (100 µmol/L) inhibited the 1.7- to 3.0-pA currents, which were restored by diazoxide (10 µmol/L, Figure 3C).
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To confirm myocardial mitoKATP activity of reconstituted channels, the effect of HMR-1098, a selective sarcKATP antagonist, was examined. HMR-1098 at a concentration up to 100 µmol/L had no significant effect on the activity of the reconstituted channels. The NPo values in the absence and presence of HMR-1098 were 0.63±0.11 and 0.67±0.11, respectively. However, the nonselective KATP antagonist glibenclamide (10 to 100 µmol/L) inhibited the activity of the reconstituted channels in a concentration-dependent manner; these channels were also reactivated by addition of 10 µmol/L diazoxide (Figure 4A). Figure 4B shows that HMR-1098 (100 µmol/L) had no effect on the 1.7- to 3.0-pA currents. However, glibenclamide (100 µmol/L) inhibited these currents, which were again restored by diazoxide (10 µmol/L, Figure 4C).
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Effect of O2-· on the Activity of Reconstituted Myocardial mitoKATP Channels
Representative recordings depicting the effect of O2-· on the activity of reconstituted mitoKATP channels are presented in Figure 5A. Addition of xanthine/xanthine oxidase (100 µmol/L per 0.038 U/mL) to the cis solution resulted in a rapid activation of the channels within 1 minute. A stacked opening of these channels was observed in xanthine/xanthine oxidase–treated patches. The NPo of the reconstituted channels was increased from 0.60±0.10 to 1.94±0.02 (Figure 5B). However, neither xanthine nor xanthine oxidase alone had an effect on channel activity. In addition, xanthine/xanthine oxidase had no significant effect on the conductance of the lipid membrane in the absence of channel protein incorporation. Figure 5C shows that xanthine/xanthine oxidase induced large conductance currents (4.0 pA). As indicated in Figure 5A, these currents represent the stacked opening of two mitoKATP channels instead of the opening of a new channel.
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Modulation of O2-· Action on Reconstituted Myocardial mitoKATP Channels by Antagonists and N-Ethylmaleimide
To explore the mechanism of action of O2-· on mitoKATP channel activity, mitoKATP antagonists and N-ethylmaleimide were used to modulate O2-·-induced activation of mitoKATP. As shown in Figure 6A, 5-HD (100 µmol/L) completely abolished xanthine/xanthine oxidase–induced activation of mitoKATP channels. The NPo values in the presence of 5-HD and the subsequent xanthine/xanthine oxidase were 0.14±0.05 and 0.14±0.05, respectively. N-Ethylmaleimide (2 mmol/L) had no significant effect on basal channel activity. However, pretreatment with N-ethylmaleimide resulted in a complete blockade of xanthine/xanthine oxidase–induced activation of mitoKATP channels. It was also found that glibenclamide (100 µmol/L) completely blocked mitoKATP activation by xanthine/xanthine oxidase. In contrast, MgATP (1 mmol/L) was less effective in blocking xanthine/xanthine oxidase–induced channel activation. The NPo in the presence of MgATP and the subsequent xanthine/xanthine oxidase was 0.10±0.08 and 1.66±0.13, respectively. Figure 6B shows that 5-HD (100 µmol/L) and glibenclamide (100 µmol/L) abolished xanthine/xanthine oxidase–activated 1.7- to 3.0-pA currents and the stacked opening of these currents. N-Ethylmaleimide (2 mmol/L) also blocked stacked opening of mitoKATP channels induced by xanthine/xanthine oxidase.
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Discussion |
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In the present study, we first reconstituted mitoKATP channels from bovine ventricular myocardium into a planar lipid bilayer and examined their electric and pharmacological properties. These channels displayed an electric rectifying property with a mean conductance of 56 pS under symmetrical KCl (150 mmol/L). The rectifying property of the channels resembled that of mitoKATP previously isolated from rat liver,25 but the channels had a larger conductance compared with the mitoKATP observed in intact liver mitoplasts (9.7 pS)4 and from rat liver (10 pS at 100 mmol/L KCl).25,26 In previous studies, the variation in mitoKATP channel conductance was assumed to be associated with the incorporation of multiple channels to form a cluster and the simultaneous switching of these channels in lipid bilayers.4,25–27 We also found that the reversal potential of these mitochondrial channels at asymmetrical KCl (150/50 mmol/L) was close to the equilibrium potential for K+, indicating that these channels are primarily permeable to K+.
In agreement with the results of previous studies,4,25–27 the mitoKATP channel was active under control conditions. In some cases, the channel was fully open. However, there is evidence that mitoKATP channels are in a closed state in vivo under physiological conditions.2 The inner mitochondrial membrane potential is estimated to be -180 mV. To prevent excessive accumulation of cations in the mitochondria, the mitoKATP channel must be finely regulated in vivo, presumably by ATP or other nucleotides, or long-chain acyl–coenzyme A (CoA) esters.28 The activity of mitoKATP channels would control the concentrations of K+ in the mitochondria and regulate the function of the mitochondria. In response to the ATP decrease, mitoKATP channels may be activated, which would consequently result in an increase in ATP production in the mitochondria.2 Therefore, the activation of mitoKATP channels may represent an important cellular adaptive mechanism. Accordingly, the high activity of mitoKATP channels observed under control conditions may be due to the absence and lack of inhibition by ATP or acyl-CoA esters in the reconstituted bilayer system.
To further characterize reconstituted mitoKATP currents, we tested the effects of different KATP antagonists and agonists on channel activity. It was found that the activity of reconstituted channels was inhibited by ATP in the presence of Mg2+ ions and reactivated by GTP, indicating that these mitochondrial channels are ATP and GTP sensitive. These results are consistent with those of previous studies using fluorescence measurements and proteoliposomes containing reconstituted mitoKATP from rat liver mitochondria.23 ATP-induced inhibition of channel activity represents an important feature of mitoKATP channels as reported previously.4,25–27 In agreement with previous studies,25,26 ATP was effective only when added to the trans side of our bilayer preparations. This indicates that the regulatory site of ATP is only on one side of the channel protein. Given the general agreement that the cis side represents the cytosolic side of reconstituted channel proteins, it is likely that ATP binds to the matrix side of mitoKATP channels in our preparations. In previous studies, using K+ flux assays in proteoliposomes containing reconstituted mitoKATP channels, however, ATP has been reported to act on the cytosolic side of the channel protein.23 We do not know why there is a difference in the location of ATP action observed in bilayer and proteoliposome studies. It is possible that mitoKATP channels are somehow incorporated into the bilayer with the ATP binding site oriented to the trans side. This controversy regarding the sidedness of mitoKATP channels in the bilayer and the location of action of the channel modulating ligands remains to be clarified.4,23
Using proteoliposomes containing reconstituted myocardial mitoKATP or sarcKATP channels or intact cardiomyocytes, diazoxide opened the mitoKATP channel with an EC50 of 3 to 27 µmol/L, which was 2000-fold less than that required to open sarcKATP channels.8,11 Similarly, 5-HD selectively inhibits the activity of mitoKATP channels but not sarcKATP channels.9,12 Using these specific inhibitors or activators of mitoKATP channels, the present study demonstrated that reconstituted mitochondrial K+ channels were also inhibited by 5-HD and activated by diazoxide. The inhibition of KATP channels by channel antagonists (ie, glibenclamide) under basal conditions and subsequent reactivation by channel openers has also been reported previously using a bilayer clamping technique.29 In some experiments, there were some remaining small conductance currents after ATP or 5-HD treatment, which was also observed previously in liver mitoKATP channels.4,25–27 The identity of these currents is currently unknown; however, they could be due to a fast flickering of mitoKATP channels, which was filtered during recording.
With regard to the inhibitory effect of 5-HD, a previous study has shown that 5-HD did not inhibit mitoKATP channels in both intact mitochondria and reconstituted proteoliposomes containing mitoKATP channels unless ATP and a channel opener (ie, diazoxide) were present.30 The reason for this discrepancy is not clear. It is possible that an important regulatory factor of mitoKATP channels targeted by 5-HD was lost during the reconstitution process in that previous study.30 It is also possible that mitoKATP channels need to be in a specific conformation or open state for 5-HD to be effective, a condition that may occur under the present experimental conditions (open actively) but not in intact and respiring mitochondria. In support of this view, glibenclamide, a structurally unrelated KATP antagonist, also inhibited mitoKATP activity under basal conditions, in which it is ineffective in the intact mitochondria.30
To exclude the possibility that the ATP-sensitive K+ current is derived from sarcKATP channels, we examined the effect of HMR-1098, a specific sarcKATP antagonist,14,31 on the activity of reconstituted channels. As expected, HMR-1098 had no significant effect on channel activity. Previous studies have shown that HMR-1098, at similar or lower concentrations, blocked the activity of sarcKATP channels.14,31 The failure of HMR-1098 to block K+ channel activity in the present study further confirms that the K+ currents recorded represent mitoKATP channels.
The present studies used submitochondrial membrane vesicles instead of purified proteins as reported previously.25–27 This experimental system is more analogous to patch-clamp studies on liver mitoplasts.4 Apart from the mitoKATP channel, we observed several other ATP-insensitive currents in some experiments, as also reported previously on mitoplasts.4 However, these channels could be generally differentiated from mitoKATP channels by their current-voltage relations, conductance, channel kinetics, or drug sensitivity. None of these ATP-insensitive channels had the characteristic rectifying properties of mitoKATP channels. A channel with a conductance of 100 pS resembles the voltage-sensitive anion-selective current recorded in liver mitoplasts.4 Another channel with a conductance of 250 pS was similar to the Ca2+-activated K+ channel recently reported in the mitochondrial inner membrane.32 Another displayed "spiky" activity and several conductance levels. Because these channels were rare and inconsistent, the present study did not characterize them.
Superoxide anions or other ROS have been reported to activate sarcKATP channels in inside-out membrane patches of ventricular myocytes.33–35 The effect of O2-· on sarcKATP channels has been implicated in myocardial ischemia/reperfusion injury. In the present study, we demonstrated that addition of xanthine/xanthine oxidase markedly increased the NPo of reconstituted mitoKATP channels, whereas xanthine or xanthine oxidase alone had no effect on the activity of these channels. The apparent increase in current amplitude (2-fold) may be due to the "stack" or simultaneous opening of two or three channels of the same type instead of an increase in channel conductance or nonspecific ion permeability, given that it can be blocked by 5-HD and glibenclamide. In addition, the activation of mitoKATP channels by O2-· could not be due to a nonspecific effect of O2-· on the lipid environment, given that xanthine/xanthine oxidase had no significant effect on the conductance of the lipid membrane in the absence of mitoKATP channel incorporation. To our knowledge, these results provide the first direct evidence that O2-· activates myocardial mitoKATP channels.
Because mitoKATP channels were reconstituted in lipid bilayers, it is possible that O2-· can directly act on the channel protein and result in channel activation. To test this hypothesis, N-ethylmaleimide, a sulfhydryl alkylating agent, was used to determine whether a modification of channel protein by sulfhydryl alkylation alters the effect of O2-· on the activity of these channels. We found that O2-·-induced channel activation was completely blocked after treatment with N-ethylmaleimide, suggesting that sulfhydryl groups in channel protein may be the target for the action of O2-·. Previous studies have shown that switching of the neighboring sulfhydryls from the oxidized to the reduced state or vice versa is able to modulate channel conformation and channel gating.36 A number of redox active compounds have been shown to modulate the activity of reconstituted mitoKATP channels from rat liver, such as p-diethylaminoethylbenzoate and pelargonidine. These compounds may act as electron donors or acceptors and target sulfhydryl groups of mitoKATP channel protein.37 Our findings further support the view that redox status may regulate the activity of mitoKATP channels in the myocardium. Furthermore, O2-·-induced activation of mitoKATP channels is substantially blocked by a specific mitoKATP channel antagonist, 5-HD. This indicates that O2-· mainly alters the gating mechanism of mitoKATP channels and that the functional activation due to modification of the channel protein by O2-· can be blocked by a selective mitoKATP inhibitor. However, the activation of mitoKATP channels induced by O2-· was not blocked by ATP, thus suggesting that O2-· activates mitoKATP channels through a different mechanism from the sensitivity of these channels to ATP.
The present study did not attempt to address the significance of O2-·-induced activation of mitoKATP channels in IPC or ischemia/reperfusion injury. ROS have been shown to serve as important intracellular signaling molecules in the activation of IPC.15–20 However, the precise intracellular mechanism for the action of ROS remains unclear. Although there is evidence suggesting that mitoKATP channel activation promotes ROS production,38 recent studies demonstrated that 5-HD did not affect the oxidant generation during preconditioning, suggesting that ROS formation during preconditioning did not likely result from mitoKATP activation.16 The results of the present study demonstrate that O2-· is a potent activator of myocardial mitoKATP channels. Therefore, ROS, such as O2-· generated during IPC, may activate mitoKATP channels, thereby leading to a cardioprotective effect.
In summary, the present study reconstituted and characterized myocardial mitoKATP channels in planar lipid bilayers. The mitoKATP channels were inhibited by MgATP and 5-HD and activated by diazoxide. O2-· significantly increased channel activity, which was associated with modification of the sulfhydryl groups of the channel protein. It is suggested that activation of myocardial mitoKATP channels by O2-· may represent an important intracellular pathway in mediating the protective effect associated with IPC and potassium channel openers.
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Acknowledgments |
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This study was supported by NIH Grants HL-57244 (to P.-L.L.), DK54927 (to A.-P.Z.), and HL-51055 (to W.B.C.) and American Heart Association Established Investigator Grant 9940167N (to P.-L.L.). We thank Gretchen Barg for secretarial assistance.
Received April 6, 2001; revision received October 31, 2001; accepted October 31, 2001.
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