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J. Biol. Chem., Vol. 276, Issue 36, 33369-33374, September 7, 2001

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Identification and Properties of a Novel Intracellular (Mitochondrial) ATP-sensitive Potassium Channel in Brain^*

Robert Bajgar, Subramaniam Seetharaman, Alicia J. Kowaltowski, Keith D. Garlid, and Petr Paucek^§

From the Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921

Received for publication, April 13, 2001, and in revised form, June 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K_ATP channel openers is known to involve the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP). Brain is also protected by ischemic preconditioning and K_ATP channel openers, and it has been suggested that mitoK_ATP may also play a key role in brain protection. However, it is not known whether mitoK_ATP exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK_ATP. We report partial purification and reconstitution of a new mitoK_ATP from rat brain mitochondria. We measured K⁺ flux in proteoliposomes and found that brain mitoK_ATP is regulated by the same ligands as those that regulate mitoK_ATP from heart and liver. We also examined the effects of opening and closing mitoK_ATP on brain mitochondrial respiration, and we estimated the amount of mitoK_ATP by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK_ATP from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK_ATP per milligram of mitochondrial protein than liver or heart.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The inner membranes of liver and heart mitochondria contain an ATP-sensitive K⁺ channel (mitoK_ATP),¹ whose regulation has been studied in both intact mitochondria and liposomes containing reconstituted, purified mitoK_ATP (1-6). MitoK_ATP is inhibited by ATP, ADP, long-chain CoA esters, glyburide, and 5-hydroxydecanoate (5-HD). The ATP-inhibited channel is opened by GTP, GDP, cromakalim, diazoxide, and other K_ATP channel openers. K_1/2 values for regulation of K⁺ flux by these ligands are virtually identical in heart and liver mitoK_ATP. The same set of ligands regulates K_ATP channels found in plasma membranes (cellK_ATP); however, in some cases the effects are different. For example, cellK_ATP is opened by ADP and long-chain CoA esters (7), whereas mitoK_ATP is blocked by these ligands (1, 4). There are also important pharmacological differences: cellK_ATP from cardiac sarcolemma is essentially insensitive to diazoxide and 5-HD, whereas mitoK_ATP is sensitive to both agents (3).

It has been known for some time that K_ATP channel openers protect the heart against ischemia-reperfusion injury and that K_ATP channel blockers prevent this protection (8-10). In a study on cardiac ischemia-reperfusion injury, we exploited the pharmacological differences between cellK_ATP and mitoK_ATP in heart to show that mitoK_ATP mediates the cardioprotective effects of K_ATP channel openers (11).

Ischemia-reperfusion injury in brain is an important medical problem. Several studies have shown that K_ATP channel openers such as cromakalim and diazoxide are protective in brain models of ischemia-reperfusion (12, 13), and Domoki et al. (14) have suggested that the mechanism of tissue protection in brain is similar to that in heart and may be mediated by the opening of mitoK_ATP. It is important, therefore, to establish whether or not brain mitochondria contain a K_ATP channel and to determine its properties and regulation.

In this work, we report that rat brain contains an active mitoK_ATP whose regulation is qualitatively identical to regulation of mitoK_ATP from heart and liver. We also observed that brain mitochondria appeared to be significantly enriched in mitoK_ATP. This was verified with a novel technique for labeling the mitochondrial sulfonylurea receptor (mitoSUR). The labeling studies indicate that brain mitochondria contain approximately seven times more mitoK_ATP per milligram of mitochondrial protein than heart and liver mitochondria.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mitochondrial Isolation-- Mitochondria were isolated by differential centrifugation from rat brain cortex (15) and liver (16). The brain mitochondrial preparation utilizes digitonin to disrupt synaptosomal vesicles and is considered to provide a population that is representative of both glial and neuronal tissue (15). Mitochondrial protein was estimated using the Biuret reaction (17).

Measurement of Mitochondrial Respiration-- Respiration was measured at 25 °C with a Clark-type oxygen electrode in K⁺- and TEA⁺-based media containing 0.5 mg of mitochondrial protein/ml, 2.77 mM CaCl₂, 1.38 mM MgCl₂, 0.5 mM dithiothreitol, 20 mM imidazole, 2 mM malate, 5 mM pyruvate, 3 mM phosphate, and 10 mM EGTA, pH 7.1 (adjusted by KOH or TEAOH).

Measurement of Mitochondrial Volume-- Changes in mitochondrial matrix volume, due to net K⁺ salt transport into mitochondria, were monitored by quantitative light scattering, as described previously (3, 5, 16). Mitochondria (0.1 mg/ml) were incubated in K⁺ salts of 135 mM chloride, 5 mM TES, 5 mM glutamate, 1 mM malate, 2.5 mM inorganic phosphate, 0.5 mM EGTA, and 0.5 mM MgCl₂, pH 7.4. A comparison of the linear osmotic responses of matrix water content, W_m, and the light scattering parameter was used to convert the values to matrix water content, as described previously (16).

Solubilization and Fractionation of the Mitochondrial K⁺ Channel-- 30 mg of rat brain mitochondria was centrifuged at 15,000 × g for 10 min, and the pellet was solubilized in 10 ml of 3% Triton X-100, 0.1% -mercaptoethanol, 0.2 mM EGTA, and 50 mM Tris-HCl, pH 7.2. After incubation on ice for 90 min, the mixture was centrifuged at 180,000 × g for 40 min. The supernatant was loaded onto a 10-ml DEAE-cellulose column pre-equilibrated with column buffer, which contained 0.5% Triton X-100, 0.1% -mercaptoethanol, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.2. The column was washed sequentially with column buffer containing 0, 100, 180, 250, and 500 mM KCl, two column bed volumes each, at 0.2 ml/min. Column eluate was continuously monitored for UV absorption and conductivity and collected in 1-ml fractions. Appropriate selected fractions were dialyzed overnight against column buffer, photolabeled by BODIPY-FL-glyburide, analyzed by SDS-PAGE, and reconstituted into liposomes for transport activity studies. Electrophoresis was carried out using 10% polyacrylamide gels (18), with gel patterns visualized by Coomassie Brilliant Blue R-250.

To further purify mitoK_ATP, the DEAE-cellulose mitoK_ATP fractions were combined (5 ml) and dialyzed overnight at 4 °C with 1 ml of ATP-agarose against column buffer containing 1 mM MgCl₂. The dialysate was poured into a small column (1 ml) and washed sequentially with the dialyzing buffer alone, buffer with 200 mM NaCl, dialyzing buffer alone, and buffer with 20 mM Tris-buffered ATP (three bed volumes each). After dialysis, the fractions eluted with ATP were reconstituted into liposomes and analyzed by SDS-PAGE.

Reconstitution of MitoK_ATP-- Reconstitution of mitoK_ATP proteins into PBFI-loaded liposomes was performed as described previously (1, 19). Internal medium contained 100 mM TEA-SO₄, 1 mM EDTA, 25 mM TEA-HEPES, pH 6.8, and 300 µM PBFI. Kinetic studies were performed in external medium containing 150 mM KCl, 1 mM EDTA, 1 mM MgCl, and 25 mM TEA-HEPES, pH 7.2, at a proteoliposome concentration of 0.4 mg of lipid/ml. K⁺ flux through mitoK_ATP was initiated by 0.5 µM CCCP, which provides charge compensation for the electrophoretic K⁺ flux. Fluorescence changes of the K⁺-sensitive probe PBFI were monitored using an SLM/Aminco 8000C fluorescence spectrophotometer (_ex/_em = 345/485 nm), with fluorescence signals calibrated to K⁺ flux as previously described (20). Results were plotted as the normalized values J/J_max, where J_max is the difference between fluxes in the absence and presence of 200 µM (saturating) ATP, and J is the difference between fluxes in the presence or absence of the mitoK_ATP modulator. K_1/2 values and Hill coefficients (n_H) were determined from three independent experiments by non-linear regression fits to sigmoidal curves using ORIGIN 6.0 software.

BODIPY-FL-Glyburide Labeling of MitoK_ATP-- DEAE-cellulose fractions containing mitoK_ATP in Triton X-100 micelles were incubated for 60 min at 25 °C with 50 nM BODIPY-FL-glyburide, in the presence or absence of 1 µM unlabeled glyburide (control). Reaction mixtures were UV-irradiated (5000 J/m²,  254 nm) for 6 min at 4 °C (21, 22) and then precipitated to remove unbound probe (23). Precipitated, delipidated proteins were dissolved with 5% SDS in 50 mM Tris-HCl, pH 6.8, then diluted 20 times with 50 mM Tris-HCl, pH 6.8, and analyzed directly for fluorescence (_ex/_em = 493/515 nm).

Chemicals-- PBFI and BODIPY-FL-glyburide were purchased from Molecular Probes; electrophoresis chemicals were obtained from Bio-Rad; column resins and other chemicals were from Sigma Chemical Co.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Reconstitution of Brain MitoK_ATP-- We reconstituted brain mitoK_ATP using protocols identical to those used for mitoK_ATP from heart and liver (1). Fig. 1A shows the reconstitutively active mitoK_ATP fraction that was eluted from a DEAE-cellulose column. This fraction contains several protein bands, including 55- and 63-kDa proteins, similar to those observed in active fractions obtained from heart or liver mitochondria. Further purification of this fraction on an ATP affinity column yielded a reconstitutively active fraction containing only 55- and 63-kDa proteins (Fig. 1B). Upon reconstitution, the proteoliposomes exhibited K⁺ flux characteristic of mitoK_ATP (Fig. 2). CCCP was required for K⁺ flux (trace a versus trace d), confirming that the flux was electrophoretic. K⁺ flux was inhibited by 200 µM ATP (trace b), and this inhibition was reversed by 50 µM cromakalim, a K_ATP opener (trace c). As previously observed with mitoK_ATP from liver, ATP did not inhibit in the absence of Mg²⁺ ion (1).

View larger version (57K): [in this window] [in a new window]   Fig. 1.   Purification of brain mitoKATP. MitoKATP was first purified on a DEAE-cellulose column and eluted with 250 mM KCl (panel A, lane 1). This fraction was further purified on an ATP-affinity column (panel B, lane 2). The reconstitutively active fraction separates on 10% SDS-PAGE as two protein bands of 63 and 55 kDa.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   K+ flux in liposomes reconstituted with brain mitoKATP. The figure contains representative traces of intraliposomal K+, determined from PBFI fluorescence versus time. Electrophoretic K+ uptake into liposomes was initiated by the addition of 0.5 µM CCCP to provide charge compensation via H+ flux. K+ flux through mitoKATP (trace a) was inhibited by 200 µM ATP (trace b), and ATP inhibition was reversed by 50 µM cromakalim (trace c). Trace d represents a control experiment in which CCCP was omitted.

Regulation of Reconstituted Brain MitoK_ATP-- Fig. 3 contains the results of experiments designed to determine the dependence of K⁺ flux on ATP () and GTP () concentrations. The K_1/2 for ATP inhibition was 43 ± 3 µM (see Table I). The K_1/2 for GTP opening in the presence of 200 µM ATP was 3.2 µM. MitoK_ATP was also released from ATP inhibition by the K_ATP channel openers diazoxide (K_1/2 = 0.78 µM) and cromakalim (K_1/2 = 11 µM) (Fig. 4). The pharmacologically open channel (in the presence of 200 µM ATP and 2 µM diazoxide) was inhibited by 5-HD (K_1/2 = 71 µM) or glyburide (K_1/2 = 56 nM) (Fig. 5). As previously observed, 5-HD did not inhibit unless Mg²⁺, ATP, and diazoxide were all present (3). These and other data are summarized in Table I. It can be seen that all mitoK_ATP modulators were effective at concentrations similar to those found for heart and liver mitoK_ATP.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   ATP and GTP regulate reconstituted mitoKATP. The normalized mitoKATP flux ratio J/Jmax, defined under "Experimental Procedures," is plotted versus concentrations of ATP () or GTP (). ATP inhibited the channel with K1/2 = 43 µM and nH = 1. GTP reversed inhibition by 200 µM ATP with K1/2 = 3.2 µM and nH = 1. These results are representative of three separate experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Opening of brain mitoKATP by diazoxide and cromakalim. The normalized mitoKATP flux ratio J/Jmax, defined under "Experimental Procedures," is plotted versus concentrations of the KATP channel openers diazoxide (, K1/2 = 0.78 µM, nH = 2) and cromakalim (, K1/2 = 11 µM, nH = 2) in the presence of 200 µM ATP. These results are representative of three separate experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Inhibition of brain mitoKATP by glyburide and 5-hydroxydecanoate. The normalized mitoKATP flux ratio J/Jmax, defined under "Experimental Procedures," is plotted versus concentrations of the KATP channel blockers glyburide (, K1/2 = 56 nM, nH = 2) and 5-hydroxydecanoate (, K1/2 = 71 µM, nH = 2) in the presence of 200 µM ATP and 2 µM diazoxide. These results are representative of three separate experiments.

Effects of MitoK_ATP Opening and Closing on Brain Mitochondrial Matrix Volume-- Matrix swelling secondary to K⁺ influx in respiring brain mitochondria was followed by light scattering (16), with the results shown in Fig. 6. There is an initial respiration-driven uptake of K⁺ salts and water, which acts to restore the matrix K⁺ that was lost during mitochondrial isolation (24). A steady-state volume is reached, which reflects a zero net flux balance between K⁺ influx and K⁺ efflux via the mitochondrial K⁺/H⁺ antiporter (25). Matrix swelling was decreased in rate and extent by 400 µM ATP (trace b), and the control fluxes were restored by addition of 10 µM diazoxide (trace c). Matrix swelling was inhibited by further addition of 2 µM glyburide (trace d). No effects of ATP, diazoxide, or glyburide were observed when Li⁺ or TEA⁺ were substituted for medium K⁺ (data not shown). Thus, these changes are specific for K⁺ and attributable to opening and closing of mitoK_ATP.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Brain mitoKATP regulates matrix volume. Time traces of matrix water content in brain mitochondria respiring in K+ medium, as described under "Experimental Procedures." K+ uptake restores the K+ lost during isolation and eventually achieves a steady-state balance between K+ influx and K+ efflux via the K+/H+ antiporter (trace a). Steady-state volume is decreased by 400 µM ATP (trace b), which inhibits mitoKATP. Volume is increased by 10 µM diazoxide in the presence of 400 µM ATP (trace c), and decreased by 2 µM glyburide in the presence of 400 µM ATP and 10 µM diazoxide (trace d). Results are representative of five separate experiments.

Effects of MitoK_ATP Opening and Closing on Brain Mitochondrial Respiration-- The effects of mitoK_ATP on mitochondrial state 2 respiration in brain mitochondria are shown in Fig. 7. Respiration was compared in K⁺ (closed bars) and TEA⁺ (open bars) media. We previously showed that opening of heart mitoK_ATP is associated with small changes in respiration that translate to a K⁺ influx of only 24-30 nmol/mg·min (26). Brain mitochondria exhibited a significantly larger change in respiration, amounting to 16-17 ng of atom O/mg·min (Fig. 7). Assuming an H⁺/O stoichiometry of 10 (27), this corresponds to 160-170 nmol of K⁺/min·mg, about seven times larger than that observed in rat heart mitochondria. Although large, this rate of K⁺ influx does not greatly depolarize the mitochondrial membrane potential. Measurements using Safranin O fluorescence (28) indicated that decreased by only 3-6 mV, which is consistent with the magnitude of the respiratory stimulation.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effects of brain mitoKATP opening/closing on mitochondrial respiration. Oxygen consumption rates of rat brain mitochondria (0.5 mg/ml) respiring in pyruvate media, described under "Experimental Procedures." Media were made up as K+ () or TEA+ () salts in the presence of no additions (control); 200 µM ATP (+ATP); 200 µM ATP and 10 µM diazoxide (+ATP +DZX +GLY); or 200 µM ATP, 10 µM diazoxide, and 1 µM glyburide (+ATP +DZX). Bars represent the mean and S.D. from three independent experiments.

Relative Abundance of Brain MitoK_ATP-- During reconstitutions of brain mitoK_ATP, we were struck by the fact that much smaller amounts of starting material were required to achieve transport rates comparable to heart and liver. Indeed, 15 mg of rat brain mitochondria yielded rates similar to rates from 100 mg of either rat liver or heart mitochondria. This observation was consistent with the ratio of K⁺ fluxes calculated from respiration rates. We decided to examine the abundance of mitoK_ATP using an independent approach. Kramer et al. (21) had shown that the -cell sulfonylurea receptor could be labeled in detergent micelles, and we applied the same approach to photoaffinity labeling of mitoK_ATP. The results of these studies are contained in Table II. MitoSUR was seven times more abundant (per milligram of mitochondrial protein) in brain than in liver, consistent with the above studies, indicating greater transport activity in brain mitochondria. In further studies being prepared for publication, we labeled the ATP column eluate with BODIPY-FL-glyburide (±1.0 µM glyburide) and fractionated the proteins by preparative SDS-PAGE. The 63-kDa protein was specifically labeled, whereas the 55-kDa protein was not labeled.²

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report identification of an ATP-sensitive K⁺ channel in rat brain mitochondria with properties similar to heart and liver mitoK_ATP (1-3). ATP inhibition is reversed by GTP, diazoxide, or cromakalim, and the open channel is inhibited by glyburide or 5-HD (Figs. 2-5). The sensitivity to sulfonylureas and the presence of two protein bands in the purified mitoK_ATP fraction (Fig. 1) imply that mitoK_ATP is a heteromultimer consisting of a 55-kDa inwardly rectifying K⁺ channel, mitoKIR (29, 30), and a 63-kDa sulfonylurea receptor, mitoSUR (10).

Participation of mitoK_ATP in regulation of matrix volume is confirmed by the effects of ATP, diazoxide, and 5-HD shown in Fig. 6. These effects are similar to those observed in heart mitochondria (26) and are thought to reflect the dynamic volume regulation mediated by the mitochondrial K⁺ cycle in vivo, as described in the legend to Fig. 8. Increased K⁺ cycling due to mitoK_ATP opening caused a moderate increase in respiration (Fig. 7), which corresponds to a K⁺ flux of 160-170 nmol/mg·min. This degree of uncoupling due to K⁺ cycling is relatively small, but it is noteworthy that it is ~7 times greater than that observed in heart or liver mitochondria (26), a difference that was confirmed by BODIPY-FL-glyburide labeling of the partially purified proteins (Table II). The basis for the higher quantity in brain is unknown.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Mitochondrial K+ cycle. Protonmotive force (p) is generated by proton ejection via the electron transport system. p =  + 2.3(RT/F)pH, where R is the gas constant, T is temperature, F is the Faraday constant, is the membrane potential (about 190 mV), and pH is the pH gradient (about 0.3 unit) across the inner membrane. drives K+ influx by diffusion ("K+ leak") and via mitoKATP. Phosphate enters by the electroneutral Pi/OH exchanger (not shown), so that net K+ transport is accompanied by anions and osmotically obligated water. The electroneutral K+/H+ antiporter is regulated on the matrix side to respond to volume changes independently of the means used to change volume (24). The K+/H+ antiporter discharges excess K+ (accompanied by Pi and water) and thereby prevents excessive swelling. We favor the idea that mitoKATP is regulated physiologically to open during states of high ATP production, which will cause to fall. Because diffusive K+ influx is exponential with (59), it is exquisitely sensitive to such fluctuations. Thus, a 30-mV decrease in will cause a 50% decrease in diffusive K+ influx, and the matrix will contract until the K+/H+ antiporter comes back into balance at a lower steady-state volume. When mitoKATP is opened in this condition, it adds a parallel conductance pathway to compensate for the lower driving force. Therefore, its role in high work states is to prevent matrix contraction caused by mild depolarization (26). As described under "Discussion," a similar role is proposed for mitoKATP opening during ischemia, which is also associated with depolarization. If ischemia occurs without warning, however, there are no endogenous mechanisms for opening mitoKATP, and severe ischemia-reperfusion injury will result.

The unitary conductance of mitoKIR is 10 pS (31), which corresponds to a turnover of 10⁸ mol of K⁺ per mol of channel protein per minute. Dividing V_max by the turnover number yields an estimate of 1.6 fmol of channel per mg of brain mitochondrial protein. If there are four mitoSUR and mitoKIR subunits per channel, and mitoK_ATP is open 50% of the time during V_max measurements, we can estimate that brain mitochondria contain about 13 fmol of mitoSUR and mitoKIR per mg of protein.

There is intense interest in understanding the mechanism of protection against ischemia-reperfusion injury. Considerable evidence suggests that heart and brain share common pathways of ischemic protection, and it is generally agreed that K_ATP channels play an important role. Thus, both tissues are protected by ischemic preconditioning in which a brief period of ischemia protects against a subsequent longer period of ischemia (32, 33), and this protection is prevented by blockers of K_ATP channels (34-36). Moreover, both tissues are protected from ischemia-reperfusion injury if they are pretreated with pharmacological openers of K_ATP channels (12, 37). For both tissues, it was initially assumed that protection was afforded exclusively by the K_ATP channel of the plasma membrane (8, 38). This assumption was shown to be incorrect in heart by the discovery that the receptor for K_ATP channel openers and blockers, which affect ischemic protection, is the mitochondrial ATP-sensitive K⁺ channel (11). Although some experiments suggest that plasma membrane K_ATP channels may also contribute to protection (39-41), the central role of mitoK_ATP is now widely accepted (10, 42-48). It is logical to predict that the same conclusion will apply to brain (14); however, definitive evidence for this hypothesis is lacking.

To understand how mitoK_ATP opening protects the ischemic cell, it is necessary to consider a complex sequence of events, beginning with how mitoK_ATP can be opened in vivo. This occurs either by administering a K_ATP channel opener or by endogenous signals that are triggered by ischemic preconditioning. We hypothesize that these signals open mitoK_ATP by phosphorylation, but there is no direct evidence for this at present. Opening mitoK_ATP will increase K⁺ influx under all conditions, but the outcome of this influx will depend on the underlying bioenergetic state of the cell. We will consider first the resting, non-ischemic cell.

When diazoxide is added to normoxic heart cells, it induces a moderate rise in mitochondrial ROS production (49, 50), a phenomenon that may arise in the following way: In isolated mitochondria, we observe increased ROS production in response to mild matrix alkalinization.³ Matrix alkalinization is a normal concomitant of mitoK_ATP opening in the cell, because uptake of P_i equivalents will always be less than uptake of K⁺, due to the disparity in their cytosolic concentrations. The increased ROS activates kinases and triggers a signaling cascade that involves protein kinase C and other kinases, one of whose targets is mitoK_ATP itself. This signaling cascade is vital for preconditioning in heart (49-54), and scavenging ROS during this period prevents diazoxide's cardioprotective effects (49, 54). After diazoxide treatment, mitoK_ATP is open, and the cell is now significantly protected from injury caused by a test ischemia.

How does an open mitoK_ATP during ischemia reduce ischemia-reperfusion injury? Mitochondrial ATP hydrolysis accounts for a sizable fraction of the loss of high energy phosphates during ischemia, and ischemic protection in the heart is accompanied by lower rates of ATP hydrolysis (55, 56). We propose that mitoK_ATP opening is responsible for this partial preservation of cytosolic ATP by a mechanism that links volume regulation, VDAC conductance state, and ATP hydrolysis. When matrix volume contracts, due to membrane depolarization, IMS will expand reciprocally. Swelling will disrupt the structure-function of the IMS, causing dissociation of mitochondrial creatine kinase from VDAC, and increasing outer membrane permeability to nucleotides, which is mediated primarily by VDAC (57, 58). In this unprotected state (ischemia, closed mitoK_ATP, open VDAC), nucleotides will equilibrate across the outer membrane, and all of the cell's ATP will be available to support ATP hydrolysis. The rate of mitochondrial ATP hydrolysis is determined by the rate of ion leaks across the inner membrane. The leaks, in turn, depend exponentially on (59), which is in equilibrium with the free energy for ATP hydrolysis, G_P. Consequently, the extent of ATP loss at any given time will depend on G_P. These interrelationships mean that the only way to reduce ionic leak during ischemia is to lower mitochondrial |G_P| to a greater extent than cytosolic |G_P|. This is not possible when VDAC is in its high conductance state. In the protected state (ischemia, open mitoK_ATP, closed VDAC), nucleotides will not equilibrate across the outer membrane, and only mitochondrial ATP can support ATP hydrolysis. This will cause decreases in mitochondrial |G_P|, , and ion leaks, and, consequently, in the rate of ATP hydrolysis.

We have evidence in support of this hypothesis. In perfused rat hearts, we have shown that the outer membrane becomes permeable to nucleotides after ischemia-reperfusion and that the normal permeability barrier is retained in hearts protected either by ischemic preconditioning (48) or diazoxide.³ In isolated rat heart mitochondria, in which respiration was inhibited to simulate ischemia, we have shown that mitoK_ATP opening with diazoxide reduced the rate of ATP hydrolysis to 50% of the control value. This effect was mimicked by moderate osmotic swelling to decrease IMS volume. When the outer membrane was broken by excessive matrix swelling, the effect disappeared, and ATP hydrolysis became independent of matrix volume. These results show that mitoK_ATP opening reduced ATP hydrolysis, that the effect was caused by changes in matrix volume, and that the effect required an intact outer membrane (60). Accordingly, we hypothesize that mitoK_ATP opening during ischemia plays an energy-sparing role and that this occurs through preservation of the structure-function of the IMS and the low conductance state of VDAC. It should be noted that energy-sparing and preservation of IMS structure may also contribute to the rapid recovery of oxidative phosphorylation that is observed in protected hearts upon reperfusion (48).

Based on the abundant evidence that mitoK_ATP plays a key role in ischemic protection in heart (10, 11, 42-48), it is logical to predict that a similar mechanism will operate in brain (14). This hypothesis can be more readily explored now that brain mitoK_ATP has been identified and its regulation partially characterized.

	FOOTNOTES

^* This research was supported in part by American Heart Association Grant 9630004N (to P. P.) and by National Institutes of Health Grant GM55324 (to K. D. G.).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.

Present address: Dept. de Bioquímica, Instituto de Química, Universidade de São Paulo 05508-900, Brazil (supported by Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico).

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, OGI School of Science and Engineering, 20000 N.W. Walker Rd., Beaverton, OR 97006-8921. Tel.: 503-748-1399; Fax: 503-748-1464; E-mail: paucek@bmb.ogi.edu.

Published, JBC Papers in Press, July 5, 2001, DOI 10.1074/jbc.M103320200

² P. Paucek, unpublished data.

³ R. Bajgar, S. Seetharaman, A. J. Kowaltowski, K. D. Garlid, and P. Paucek, unpublished data.

	ABBREVIATIONS

The abbreviations used are: mitoK_ATP, mitochondrial ATP-sensitive K⁺ channel; cellK_ATP, plasma membrane ATP-sensitive K⁺ channel; KIR, inwardly rectifying K⁺ channel; 5-HD, 5-hydroxydecanoate; CCCP, carbonyl cyanide m-chlorophenyl; SUR, sulfonylurea receptor; IMS, mitochondrial intermembrane space; PBFI, potassium-binding benzofuran isophthalate; ROS, reactive oxygen species; TEA, tetraethylammonium; VDAC, voltage-dependent anion channel; , electrical membrane potential; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; BODIPY-FL-glyburide, green fluorescence probe BODIPY-FL-glyburide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				M. E. Pamenter, D. S.-H. Shin, M. Cooray, and L. T. Buck Mitochondrial ATP-sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western painted turtle J. Physiol., February 15, 2008; 586(4): 1043 - 1058. [Abstract] [Full Text] [PDF]

				T. P. Obrenovitch Molecular Physiology of Preconditioning-Induced Brain Tolerance to Ischemia Physiol Rev, January 1, 2008; 88(1): 211 - 247. [Abstract] [Full Text] [PDF]

				M. Zhou, H.-J. He, R. Suzuki, K.-X. Liu, O. Tanaka, M. Sekiguchi, H. Itoh, K. Kawahara, and H. Abe Localization of Sulfonylurea Receptor Subunits, SUR2A and SUR2B, in Rat Heart J. Histochem. Cytochem., August 1, 2007; 55(8): 795 - 804. [Abstract] [Full Text] [PDF]

				A. Kicinska, A. Swida, P. Bednarczyk, I. Koszela-Piotrowska, K. Choma, K. Dolowy, A. Szewczyk, and W. Jarmuszkiewicz ATP-sensitive Potassium Channel in Mitochondria of the Eukaryotic Microorganism Acanthamoeba castellanii J. Biol. Chem., June 15, 2007; 282(24): 17433 - 17441. [Abstract] [Full Text] [PDF]

				M. Ljubkovic, J. Marinovic, A. Fuchs, Z. J. Bosnjak, and M. Bienengraeber Targeted expression of Kir6.2 in mitochondria confers protection against hypoxic stress J. Physiol., November 15, 2006; 577(1): 17 - 29. [Abstract] [Full Text] [PDF]

				M. Jaburek, A. D.T. Costa, J. R. Burton, C. L. Costa, and K. D. Garlid Mitochondrial PKC{epsilon} and Mitochondrial ATP-Sensitive K+ Channel Copurify and Coreconstitute to Form a Functioning Signaling Module in Proteoliposomes Circ. Res., October 13, 2006; 99(8): 878 - 883. [Abstract] [Full Text] [PDF]

				S. Roth, J. C. Dreixler, A. R. Shaikh, K. H. Lee, and V. Bindokas Mitochondrial Potassium ATP Channels and Retinal Ischemic Preconditioning Invest. Ophthalmol. Vis. Sci., May 1, 2006; 47(5): 2114 - 2124. [Abstract] [Full Text] [PDF]

				M. T. Jiang, M. Ljubkovic, Y. Nakae, Y. Shi, W.-M. Kwok, D. F. Stowe, and Z. J. Bosnjak Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1770 - H1776. [Abstract] [Full Text] [PDF]

				H.-S. Sun, Z.-P. Feng, T. Miki, S. Seino, and R. J. French Enhanced Neuronal Damage After Ischemic Insults in Mice Lacking Kir6.2-Containing ATP-Sensitive K+ Channels J Neurophysiol, April 1, 2006; 95(4): 2590 - 2601. [Abstract] [Full Text] [PDF]

				A. D. T. Costa, C. L. Quinlan, A. Andrukhiv, I. C. West, M. Jaburek, and K. D. Garlid The direct physiological effects of mitoKATP opening on heart mitochondria Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H406 - H415. [Abstract] [Full Text] [PDF]

T. Brustovetsky, N. Shalbuyeva, and N. Brustovetsky Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain nonsynaptosomal mitochondria J. Physiol., October 1, 2005; 568(1): 47 - 59.