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• W2045201746 abstract "Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and KATP channel openers is known to involve the mitochondrial ATP-sensitive K+ channel (mitoKATP). Brain is also protected by ischemic preconditioning and KATP channel openers, and it has been suggested that mitoKATP may also play a key role in brain protection. However, it is not known whether mitoKATP exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoKATP. We report partial purification and reconstitution of a new mitoKATP from rat brain mitochondria. We measured K+ flux in proteoliposomes and found that brain mitoKATP is regulated by the same ligands as those that regulate mitoKATP from heart and liver. We also examined the effects of opening and closing mitoKATP on brain mitochondrial respiration, and we estimated the amount of mitoKATP by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoKATP from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoKATP per milligram of mitochondrial protein than liver or heart. Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and KATP channel openers is known to involve the mitochondrial ATP-sensitive K+ channel (mitoKATP). Brain is also protected by ischemic preconditioning and KATP channel openers, and it has been suggested that mitoKATP may also play a key role in brain protection. However, it is not known whether mitoKATP exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoKATP. We report partial purification and reconstitution of a new mitoKATP from rat brain mitochondria. We measured K+ flux in proteoliposomes and found that brain mitoKATP is regulated by the same ligands as those that regulate mitoKATP from heart and liver. We also examined the effects of opening and closing mitoKATP on brain mitochondrial respiration, and we estimated the amount of mitoKATP by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoKATP from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoKATP per milligram of mitochondrial protein than liver or heart. mitochondrial ATP-sensitive K+channel plasma membrane ATP-sensitive K+ channel inwardly rectifying K+channel 5-hydroxydecanoate carbonyl cyanidem-chlorophenyl sulfonylurea receptor mitochondrial intermembrane space potassium-binding benzofuran isophthalate reactive oxygen species tetraethylammonium voltage-dependent anion channel electrical membrane potential 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid polyacrylamide gel electrophoresis green fluorescence probe BODIPY-FL-glyburide The inner membranes of liver and heart mitochondria contain an ATP-sensitive K+ channel (mitoKATP),1whose regulation has been studied in both intact mitochondria and liposomes containing reconstituted, purified mitoKATP(1Paucek P. Mironova G. Mahdi F. Beavis A.D. Woldegiorgis G. Garlid K.D. J. Biol. Chem. 1992; 267: 26062-26069Abstract Full Text PDF PubMed Google Scholar, 2Garlid K.D. Paucek P. Yarov-Yarovoy V. Sun X. Schindler P.A. J. Biol. Chem. 1996; 271: 8796-8799Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 3Jaburek M. Yarov-Yarovoy V. Paucek P. Garlid K.D. J. Biol. Chem. 1998; 273: 13578-13582Abstract Full Text Full Text PDF PubMed Google Scholar, 4Paucek P. Yarov-Yarovoy V. Sun X. Garlid K.D. J. Biol. Chem. 1996; 271: 32084-32088Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Beavis A.D. Lu Y. Garlid K.D. J. Biol. Chem. 1993; 268: 997-1004Abstract Full Text PDF PubMed Google Scholar, 6Yarov-Yarovoy V. Paucek P. Jaburek M. Garlid K.D. Biochim. Biophys. Acta. 1997; 1321: 128-136Crossref PubMed Scopus (57) Google Scholar). MitoKATP 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 KATP channel openers. K12 values for regulation of K+ flux by these ligands are virtually identical in heart and liver mitoKATP. The same set of ligands regulates KATP channels found in plasma membranes (cellKATP); however, in some cases the effects are different. For example, cellKATP is opened by ADP and long-chain CoA esters (7Branstrom R. Corkey B.E. Berggren P.O. Larsson O. J. Biol. Chem. 1997; 272: 17390-17394Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), whereas mitoKATP is blocked by these ligands (1Paucek P. Mironova G. Mahdi F. Beavis A.D. Woldegiorgis G. Garlid K.D. J. Biol. Chem. 1992; 267: 26062-26069Abstract Full Text PDF PubMed Google Scholar, 4Paucek P. Yarov-Yarovoy V. Sun X. Garlid K.D. J. Biol. Chem. 1996; 271: 32084-32088Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). There are also important pharmacological differences: cellKATP from cardiac sarcolemma is essentially insensitive to diazoxide and 5-HD, whereas mitoKATP is sensitive to both agents (3Jaburek M. Yarov-Yarovoy V. Paucek P. Garlid K.D. J. Biol. Chem. 1998; 273: 13578-13582Abstract Full Text Full Text PDF PubMed Google Scholar). It has been known for some time that KATP channel openers protect the heart against ischemia-reperfusion injury and that KATP channel blockers prevent this protection (8Grover G.J. Cardiovasc. Res. 1994; 28: 778-782Crossref PubMed Scopus (67) Google Scholar, 9Grover G.J. Can. J. Physiol. Pharmacol. 1997; 75: 309-315Crossref PubMed Scopus (109) Google Scholar, 10Grover G.J. Garlid K.D. J. Mol. Cell. Cardiol. 2000; 32: 677-695Abstract Full Text PDF PubMed Scopus (384) Google Scholar). In a study on cardiac ischemia-reperfusion injury, we exploited the pharmacological differences between cellKATP and mitoKATP in heart to show that mitoKATPmediates the cardioprotective effects of KATP channel openers (11Garlid K.D. Paucek P. Yarov-Yarovoy V. Murray H.N. Darbenzio R.B. D'Alonzo A.J. Lodge N.J. Smith M.A. Grover G.J. Circ. Res. 1997; 81: 1072-1082Crossref PubMed Scopus (952) Google Scholar). Ischemia-reperfusion injury in brain is an important medical problem. Several studies have shown that KATP channel openers such as cromakalim and diazoxide are protective in brain models of ischemia-reperfusion (12Heurteaux C. Bertaina V. Widmann C. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9431-9435Crossref PubMed Scopus (186) Google Scholar, 13Reshef A. Sperling O. Zoref-Shani E. Neurosci. Lett. 1998; 250: 111-114Crossref PubMed Scopus (51) Google Scholar), and Domoki et al. (14Domoki F. Perciaccante J.V. Veltkamp R. Bari F. Busija D.W. Stroke. 1999; 30 (; discussion 2718–2719): 2713-2718Crossref PubMed Scopus (103) Google Scholar) have suggested that the mechanism of tissue protection in brain is similar to that in heart and may be mediated by the opening of mitoKATP. It is important, therefore, to establish whether or not brain mitochondria contain a KATP channel and to determine its properties and regulation. In this work, we report that rat brain contains an active mitoKATP whose regulation is qualitatively identical to regulation of mitoKATP from heart and liver. We also observed that brain mitochondria appeared to be significantly enriched in mitoKATP. 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 mitoKATP per milligram of mitochondrial protein than heart and liver mitochondria. Mitochondria were isolated by differential centrifugation from rat brain cortex (15Rosenthal R.E. Hamud F. Fiskum G. Varghese P.J. Sharpe S. J. Cereb. Blood Flow Metab. 1987; 7: 752-758Crossref PubMed Scopus (232) Google Scholar) and liver (16Beavis A.D. Brannan R.D. Garlid K.D. J. Biol. Chem. 1985; 260: 13424-13433Abstract Full Text PDF PubMed Google Scholar). 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 (15Rosenthal R.E. Hamud F. Fiskum G. Varghese P.J. Sharpe S. J. Cereb. Blood Flow Metab. 1987; 7: 752-758Crossref PubMed Scopus (232) Google Scholar). Mitochondrial protein was estimated using the Biuret reaction (17Sols A. Nature. 1947; 160: 89Crossref Scopus (7) Google Scholar). 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 mmpyruvate, 3 mm phosphate, and 10 mm EGTA, pH 7.1 (adjusted by KOH or TEAOH). Changes in mitochondrial matrix volume, due to net K+ salt transport into mitochondria, were monitored by quantitative light scattering, as described previously (3Jaburek M. Yarov-Yarovoy V. Paucek P. Garlid K.D. J. Biol. Chem. 1998; 273: 13578-13582Abstract Full Text Full Text PDF PubMed Google Scholar, 5Beavis A.D. Lu Y. Garlid K.D. J. Biol. Chem. 1993; 268: 997-1004Abstract Full Text PDF PubMed Google Scholar, 16Beavis A.D. Brannan R.D. Garlid K.D. J. Biol. Chem. 1985; 260: 13424-13433Abstract Full Text PDF PubMed Google Scholar). Mitochondria (0.1 mg/ml) were incubated in K+ salts of 135 mm chloride, 5 mm TES, 5 mm glutamate, 1 mmmalate, 2.5 mm inorganic phosphate, 0.5 mmEGTA, and 0.5 mm MgCl2, pH 7.4. A comparison of the linear osmotic responses of matrix water content,Wm, and the light scattering parameter was used to convert the values to matrix water content, as described previously (16Beavis A.D. Brannan R.D. Garlid K.D. J. Biol. Chem. 1985; 260: 13424-13433Abstract Full Text PDF PubMed Google Scholar). 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% β-mercaptoethanol, 0.2 mm EGTA, and 50 mmTris-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% β-mercaptoethanol, 1 mm EDTA, and 50 mmTris-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 againstcolumn 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 (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar), with gel patterns visualized by Coomassie Brilliant Blue R-250. To further purify mitoKATP, the DEAE-cellulose mitoKATP 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 mitoKATP proteins into PBFI-loaded liposomes was performed as described previously (1Paucek P. Mironova G. Mahdi F. Beavis A.D. Woldegiorgis G. Garlid K.D. J. Biol. Chem. 1992; 267: 26062-26069Abstract Full Text PDF PubMed Google Scholar, 19Garlid K.D. Sun X. Paucek P. Woldegiorgis G. Methods Enzymol. 1995; 260: 331-348Crossref PubMed Scopus (43) Google Scholar). 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 mitoKATP 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 (λex/λem = 345/485 nm), with fluorescence signals calibrated to K+ flux as previously described (20Garlid K.D. Shariat-Madar Z. Nath S. Jezek P. J. Biol. Chem. 1991; 266: 6518-6523Abstract Full Text PDF PubMed Google Scholar). Results were plotted as the normalized values ΔJ/ΔJmax, where ΔJmax is the difference between fluxes in the absence and presence of 200 μm (saturating) ATP, and ΔJ is the difference between fluxes in the presence or absence of the mitoKATP modulator. K12values and Hill coefficients (nH) were determined from three independent experiments by non-linear regression fits to sigmoidal curves using ORIGIN 6.0 software. DEAE-cellulose fractions containing mitoKATP 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, λ 254 nm) for 6 min at 4 °C (21Kramer W. Muller G. Girbig F. Gutjahr U. Kowalewski S. Hartz D. Summ H.D. Biochim. Biophys. Acta. 1994; 1191: 278-290Crossref PubMed Scopus (68) Google Scholar, 22Nelson D.A. Aguilar-Bryan L. Bryan J. J. Biol. Chem. 1992; 267: 14928-14933Abstract Full Text PDF PubMed Google Scholar) and then precipitated to remove unbound probe (23Wessel D. Flügge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3170) Google Scholar). 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 (λex/λem = 493/515 nm). 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. We reconstituted brain mitoKATP using protocols identical to those used for mitoKATP from heart and liver (1Paucek P. Mironova G. Mahdi F. Beavis A.D. Woldegiorgis G. Garlid K.D. J. Biol. Chem. 1992; 267: 26062-26069Abstract Full Text PDF PubMed Google Scholar). Fig.1 A shows the reconstitutively active mitoKATP 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. 1 B). Upon reconstitution, the proteoliposomes exhibited K+ flux characteristic of mitoKATP (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 KATP opener (trace c). As previously observed with mitoKATPfrom liver, ATP did not inhibit in the absence of Mg2+ ion (1Paucek P. Mironova G. Mahdi F. Beavis A.D. Woldegiorgis G. Garlid K.D. J. Biol. Chem. 1992; 267: 26062-26069Abstract Full Text PDF PubMed Google Scholar).Figure 2K+ 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig.3 contains the results of experiments designed to determine the dependence of K+ flux on ATP (●) and GTP (○) concentrations. The K12 for ATP inhibition was 43 ± 3 μm (see TableI). The K12 for GTP opening in the presence of 200 μm ATP was 3.2 μm. MitoKATP was also released from ATP inhibition by the KATP channel openers diazoxide (K12 = 0.78 μm) and cromakalim (K12 = 11 μm) (Fig.4). The pharmacologically open channel (in the presence of 200 μm ATP and 2 μmdiazoxide) was inhibited by 5-HD (K12 = 71 μm) or glyburide (K12 = 56 nm) (Fig. 5). As previously observed, 5-HD did not inhibit unless Mg2+, ATP, and diazoxide were all present (3Jaburek M. Yarov-Yarovoy V. Paucek P. Garlid K.D. J. Biol. Chem. 1998; 273: 13578-13582Abstract Full Text Full Text PDF PubMed Google Scholar). These and other data are summarized in Table I. It can be seen that all mitoKATP modulators were effective at concentrations similar to those found for heart and liver mitoKATP.Table IComparison of mitoKATP kinetic parameters among liver, heart and brainModulatorEffectK1/2BrainLiverHeartATPInhibition43.0 ± 3.0 μm43.0 μm (1)20.0 μmGTPOpening3.2 ± 0.2 μm6.9 μm (4)4.0 μmDiazoxideOpening780.0 ± 20.0 nm370.0 nm (2)490.0 nm (11)CromakalimOpening11.0 ± 2.0 μm1.0 μm (2)1.1 μm (11)GlyburideInhibition56.0 ± 5.0 nm62.0 nm (1)56.0 nm (11)5-HDInhibition71.0 ± 2.0 μm85.0 μm (3)83.0 μm (11)Data are compared from rat brain mitoKATP (n = 3) with data obtained from heart and liver mitoKATP. The former data were obtained from experiments such as those contained in Figs.Figure 3, Figure 4, Figure 5, Figure 6. The latter data were previously published (references are in parentheses). Open table in a new tab Figure 4Opening of brain mitoKATP by diazoxide and cromakalim. The normalized mitoKATP flux ratio ΔJ/ΔJmax, defined under “Experimental Procedures,” is plotted versusconcentrations of the KATP channel openers diazoxide (●,K12 = 0.78 μm,nH = 2) and cromakalim (○,K12 = 11 μm,nH = 2) in the presence of 200 μmATP. These results are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Inhibition of brain mitoKATP by glyburide and 5-hydroxydecanoate. The normalized mitoKATP flux ratio ΔJ/ΔJmax, defined under “Experimental Procedures,” is plotted versusconcentrations of the KATP channel blockers glyburide (●,K12 = 56 nm, nH= 2) and 5-hydroxydecanoate (○, K12 = 71 μm, nH = 2) in the presence of 200 μm ATP and 2 μm diazoxide. These results are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data are compared from rat brain mitoKATP (n = 3) with data obtained from heart and liver mitoKATP. The former data were obtained from experiments such as those contained in Figs.Figure 3, Figure 4, Figure 5, Figure 6. The latter data were previously published (references are in parentheses). Matrix swelling secondary to K+ influx in respiring brain mitochondria was followed by light scattering (16Beavis A.D. Brannan R.D. Garlid K.D. J. Biol. Chem. 1985; 260: 13424-13433Abstract Full Text PDF PubMed Google Scholar), 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 (24Garlid K.D. Lemasters J.J. Hackenbrock C.R. Thurman R.G. Westerhoff H.V. Integration of Mitochondrial Function: Mitochondrial Volume Control. Plenum Publishing Corp., New York1988: 257-276Google Scholar). 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 (25Garlid K.D. Biochim. Biophys. Acta. 1996; 1275: 123-126Crossref PubMed Scopus (190) Google Scholar). 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 mitoKATP. The effects of mitoKATP 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 mitoKATP is associated with small changes in respiration that translate to a K+ influx of only 24–30 nmol/mg·min (26Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H649-H657Crossref PubMed Google Scholar). 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 (28Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar) indicated that ΔΨ decreased by only 3–6 mV, which is consistent with the magnitude of the respiratory stimulation. During reconstitutions of brain mitoKATP, 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 mitoKATPusing an independent approach. Kramer et al. (21Kramer W. Muller G. Girbig F. Gutjahr U. Kowalewski S. Hartz D. Summ H.D. Biochim. Biophys. Acta. 1994; 1191: 278-290Crossref PubMed Scopus (68) Google Scholar) had shown that the β-cell sulfonylurea receptor could be labeled in detergent micelles, and we applied the same approach to photoaffinity labeling of mitoKATP. 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. 2P. Paucek, unpublished data.Table IIRelative abundance of mitoKATP in brain and liver mitochondriaLiverBrainRatioStarting protein content92.4 mg9.8 mg9.4 :1BODIPY-FL-glyburide fluorescence (arbitrary units)2044 ± 3431457 ± 2241 :1.4BODIPY-FL-glyburide fluorescence/starting protein content22 ± 3149 ± 221 :7MitoKATP was covalently labeled in detergent micelles by BODIPY-FL-glyburide, as described in text, and the fluorescence was normalized to starting protein (n = 3). Open table in a new tab MitoKATP was covalently labeled in detergent micelles by BODIPY-FL-glyburide, as described in text, and the fluorescence was normalized to starting protein (n = 3). We report identification of an ATP-sensitive K+channel in rat brain mitochondria with properties similar to heart and liver mitoKATP (1Paucek P. Mironova G. Mahdi F. Beavis A.D. Woldegiorgis G. Garlid K.D. J. Biol. Chem. 1992; 267: 26062-26069Abstract Full Text PDF PubMed Google Scholar, 2Garlid K.D. Paucek P. Yarov-Yarovoy V. Sun X. Schindler P.A. J. Biol. Chem. 1996; 271: 8796-8799Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 3Jaburek M. Yarov-Yarovoy V. Paucek P. Garlid K.D. J. Biol. Chem. 1998; 273: 13578-13582Abstract Full Text Full Text PDF PubMed Google Scholar). ATP inhibition is reversed by GTP, diazoxide, or cromakalim, and the open channel is inhibited by glyburide or 5-HD (Figs. Figure 2, Figure 3, Figure 4, Figure 5). The sensitivity to sulfonylureas and the presence of two protein bands in the purified mitoKATPfraction (Fig. 1) imply that mitoKATP is a heteromultimer consisting of a 55-kDa inwardly rectifying K+ channel, mitoKIR (29Mironova G.D. Fedotcheva N.I. Makarov P.R. Pronevich L.A. Mironov G.P. Biofizika. 1981; 26: 451-457PubMed Google Scholar, 30Mironova G.D. Skarga Y.Y. Grigoriev S.M. Yarov-Yarovoy V.M. Alexandrov A.V. Kolomytkin O.V. Membr. Cell Biol. 1996; 10: 429-437Google Scholar), and a 63-kDa sulfonylurea receptor, mitoSUR (10Grover G.J. Garlid K.D. J. Mol. Cell. Cardiol. 2000; 32: 677-695Abstract Full Text PDF PubMed Scopus (384) Google Scholar). Participation of mitoKATP 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 (26Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H649-H657Crossref PubMed Google Scholar) 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 mitoKATP 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 (26Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H649-H657Crossref PubMed Google Scholar), a difference that was confirmed by BODIPY-FL-glyburide labeling of the partially purified proteins (TableII). The basis for the higher quantity in brain is unknown. The unitary conductance of mitoKIR is 10 pS (31Mironova G.D. Grigoriev S.M. Skarga Y.Y. Negoda A.E. Kolomytkin O.V. Membr. Cell Biol. 1997; 10: 583-591PubMed Google Scholar), which corresponds to a turnover of 108 mol of K+ per mol of channel protein per minute. Dividing Vmax 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 mitoKATP is open 50% of the time during Vmax 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 KATP 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 (32Murry C.E. Richard V.J. Reimer K.A. Jennings R.B. Circ. Res. 1990; 66: 913-931Crossref PubMed Scopus (752) Google Scholar, 33Heurteaux C. Lauritzen I. Widmann C. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4666-4670Crossref PubMed Scopus (542) Google Scholar), and this protection is prevented by blockers of KATP channels (34Auchampach J.A. Grover G.J. Gross G.J. Cardiovasc. Res. 1992; 26: 1054-1062Crossref PubMed Scopus (263) Google Scholar, 35Hide E.J. Thiemermann C. Cardiovasc. Res. 1996; 31: 941-946Crossref PubMed Scopus (75) Google Scholar, 36Perez-Pinzon M.A. Born J.G. Neuroscience. 1999; 89: 453-459Crossref PubMed Scopus (121) Google Scholar). Moreover, both tissues are protected from ischemia-reperfusion injury if they are pretreated with pharmacological openers of KATP channels (12Heurteaux C. Bertaina V. Widmann C. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9431-9435Crossref PubMed Scopus (186) Google Scholar, 37Grover G.J. McCullough J.R. Henry D.E. Conder M.L. Sleph P.G. J. Pharmacol. Exp. Ther. 1989; 251: 98-104PubMed Google Scholar). For both tissues, it was initially assumed that protection was afforded exclusively by the KATP channel of the plasma membrane (8Grover G.J. Cardiovasc. Res. 1994; 28: 778-782Crossref PubMed Scopus (67) Google Scholar,38Wind T. Prehn J.H. Peruche B. Krieglstein J. Brain Res. 1997; 751: 295-299Crossref PubMed Scopus (39) Google Scholar). This assumption was shown to be incorrect in heart by the discovery that the receptor for KATP channel openers and blockers, which affect ischemic protection, is the mitochondrial ATP-sensitive K+ channel (11Garlid K.D. Paucek P. Yarov-Yarovoy V. Murray H.N. Darbenzio R.B. D'Alonzo A.J. Lodge N.J. Smith M.A. Grover G.J. Circ. Res. 1997; 81: 1072-1082Crossref PubMed Scopus (952) Google Scholar). Although some experiments suggest that plasma membrane KATP channels may also contribute to protection (39D'Hahan N. Moreau C. Prost A.L. Jacquet H. Alekseev A.E. Terzic A. Vivaudou M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12162-12167Crossref PubMed Scopus (166) Google Scholar, 40Toyoda Y. Friehs I. Parker R.A. Levitsky S. McCully J.D. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H2694-H2703Crossref PubMed Google Scholar, 41Sanada S. Kitakaze M. Asanuma H. Harada K. Ogita H. Node K. Takashima S. Sakata Y. Asakura M. Shinozaki Y. Mori H. Kuzuya T. Hori M. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H256-H263Crossref PubMed Google Scholar), the central role of mitoKATP is now widely accepted (10Grover G.J. Garlid K.D. J. Mol. Cell. Cardiol. 2000; 32: 677-695Abstract Full Text PDF PubMed Scopus (384) Google Scholar, 42Hu H. Sato T. Seharaseyon J. Liu Y. Johns D.C. O'Rourke B. Marbán E. Mol. Pharmacol. 1999; 55: 1000-1005Crossref PubMed Scopus (127) Google Scholar, 43Liu Y. Sato T. O'Rourke B. Marbán E. Circulation. 1998; 97: 2463-2469Crossref PubMed Scopus (735) Google Scholar, 44Sato T. O'Rourke B. Marbán E. Circ. Res. 1998; 83: 110-114Crossref PubMed Scopus (335) Google Scholar, 45Gross G.J. Fryer R.M. Circ. Res. 1999; 84: 973-979Crossref PubMed Scopus (454) Google Scholar, 46Szewczyk A. Marbán E. Trends Pharmacol. Sci. 1999; 20: 157-161Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 47Takeo S. Nasa Y. Cardiovasc. Res. 1999; 43: 32-43Crossref PubMed Scopus (34) Google Scholar, 48Laclau M.N. Boudina S. Thambo J.B. Tariosse L. Gouverneur G. Bonoron-Adele S. Saks V.A. Garlid K.D. Dos Santos P. J. Mol. Cell. Cardiol. 2001; 33: 947-956Abstract Full Text PDF PubMed Scopus (93) Google Scholar). It is logical to predict that the same conclusion will apply to brain (14Domoki F. Perciaccante J.V. Veltkamp R. Bari F. Busija D.W. Stroke. 1999; 30 (; discussion 2718–2719): 2713-2718Crossref PubMed Scopus (103) Google Scholar); however, definitive evidence for this hypothesis is lacking. To understand how mitoKATP opening protects the ischemic cell, it is necessary to consider a complex sequence of events, beginning with how mitoKATP can be opened in vivo. This occurs either by administering a KATPchannel opener or by endogenous signals that are triggered by ischemic preconditioning. We hypothesize that these signals open mitoKATP by phosphorylation, but there is no direct evidence for this at present. Opening mitoKATP 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 (49Pain T. Yang X.M. Critz S.D. Yue Y. Nakano A. Liu G.S. Heusch G. Cohen M.V. Downey J.M. Circ. Res. 2000; 87: 460-466Crossref PubMed Scopus (616) Google Scholar, 50Forbes R.A. Steenbergen C. Murphy E. Circ. Res. 2001; 88: 802-809Crossref PubMed Scopus (356) Google Scholar), a phenomenon that may arise in the following way: In isolated mitochondria, we observe increased ROS production in response to mild matrix alkalinization. 3R. Bajgar, S. Seetharaman, A. J. Kowaltowski, K. D. Garlid, and P. Paucek, unpublished data. Matrix alkalinization is a normal concomitant of mitoKATP opening in the cell, because uptake of Pi 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 mitoKATP itself. This signaling cascade is vital for preconditioning in heart (49Pain T. Yang X.M. Critz S.D. Yue Y. Nakano A. Liu G.S. Heusch G. Cohen M.V. Downey J.M. Circ. Res. 2000; 87: 460-466Crossref PubMed Scopus (616) Google Scholar, 50Forbes R.A. Steenbergen C. Murphy E. Circ. Res. 2001; 88: 802-809Crossref PubMed Scopus (356) Google Scholar, 51Downey J.M. Cohen M.V. Adv. Exp. Med. Biol. 1997; 430: 39-55Crossref PubMed Scopus (77) Google Scholar, 52Liu G.S. Cohen M.V. Mochly-Rosen D. Downey J.M. J. Mol. Cell. Cardiol. 1999; 31: 1937-1948Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 53Dorn 2nd, G.W. Souroujon M.C. Liron T. Chen C.H. Gray M.O. Zhou H.Z. Csukai M. Wu G. Lorenz J.N. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12798-12803Crossref PubMed Scopus (324) Google Scholar, 54Wang S. Cone J. Liu Y. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H246-H255Crossref PubMed Google Scholar), and scavenging ROS during this period prevents diazoxide's cardioprotective effects (49Pain T. Yang X.M. Critz S.D. Yue Y. Nakano A. Liu G.S. Heusch G. Cohen M.V. Downey J.M. Circ. Res. 2000; 87: 460-466Crossref PubMed Scopus (616) Google Scholar, 54Wang S. Cone J. Liu Y. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H246-H255Crossref PubMed Google Scholar). After diazoxide treatment, mitoKATP is open, and the cell is now significantly protected from injury caused by a test ischemia. How does an open mitoKATP 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 (55McPherson C.D. Pierce G.N. Cole W.C. Am. J. Physiol. 1993; 265: H1809-H1818PubMed Google Scholar, 56Grover G.J. Newburger J. Sleph P.G. Dzwonczyk S. Taylor S.C. Ahmed S.Z. Atwal K.S. J. Pharmacol. Exp. Ther. 1991; 257: 156-162PubMed Google Scholar). We propose that mitoKATPopening 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 (57Rostovtseva T. Colombini M. Biophys. J. 1997; 72: 1954-1962Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 58Lee A.C. Xu X. Blachly-Dyson E. Forte M. Colombini M. J. Membr. Biol. 1998; 161: 173-181Crossref PubMed Scopus (140) Google Scholar). In this unprotected state (ischemia, closed mitoKATP, 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 ΔΨ (59Garlid K.D. Beavis A.D. Ratkje S.K. Biochim. Biophys. Acta. 1989; 976: 109-120Crossref PubMed Scopus (88) Google Scholar), which is in equilibrium with the free energy for ATP hydrolysis, ΔGP. Consequently, the extent of ATP loss at any given time will depend on ΔGP. These interrelationships mean that the only way to reduce ionic leak during ischemia is to lower mitochondrial ‖ΔGP‖ to a greater extent than cytosolic ‖ΔGP‖. This is not possible when VDAC is in its high conductance state. In the protected state (ischemia, open mitoKATP, closed VDAC), nucleotides will not equilibrate across the outer membrane, and only mitochondrial ATP can support ATP hydrolysis. This will cause decreases in mitochondrial ‖ΔGP‖, ΔΨ, 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 (48Laclau M.N. Boudina S. Thambo J.B. Tariosse L. Gouverneur G. Bonoron-Adele S. Saks V.A. Garlid K.D. Dos Santos P. J. Mol. Cell. Cardiol. 2001; 33: 947-956Abstract Full Text PDF PubMed Scopus (93) Google Scholar) or diazoxide.3 In isolated rat heart mitochondria, in which respiration was inhibited to simulate ischemia, we have shown that mitoKATP 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 mitoKATP opening reduced ATP hydrolysis, that the effect was caused by changes in matrix volume, and that the effect required an intact outer membrane (60Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Biophys. J. 2001; 80: 498aAbstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Accordingly, we hypothesize that mitoKATP 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 (48Laclau M.N. Boudina S. Thambo J.B. Tariosse L. Gouverneur G. Bonoron-Adele S. Saks V.A. Garlid K.D. Dos Santos P. J. Mol. Cell. Cardiol. 2001; 33: 947-956Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Based on the abundant evidence that mitoKATP plays a key role in ischemic protection in heart (10Grover G.J. Garlid K.D. J. Mol. Cell. Cardiol. 2000; 32: 677-695Abstract Full Text PDF PubMed Scopus (384) Google Scholar, 11Garlid K.D. Paucek P. Yarov-Yarovoy V. Murray H.N. Darbenzio R.B. D'Alonzo A.J. Lodge N.J. Smith M.A. Grover G.J. Circ. Res. 1997; 81: 1072-1082Crossref PubMed Scopus (952) Google Scholar, 42Hu H. Sato T. Seharaseyon J. Liu Y. Johns D.C. O'Rourke B. Marbán E. Mol. Pharmacol. 1999; 55: 1000-1005Crossref PubMed Scopus (127) Google Scholar, 43Liu Y. Sato T. O'Rourke B. Marbán E. Circulation. 1998; 97: 2463-2469Crossref PubMed Scopus (735) Google Scholar, 44Sato T. O'Rourke B. Marbán E. Circ. Res. 1998; 83: 110-114Crossref PubMed Scopus (335) Google Scholar, 45Gross G.J. Fryer R.M. Circ. Res. 1999; 84: 973-979Crossref PubMed Scopus (454) Google Scholar, 46Szewczyk A. Marbán E. Trends Pharmacol. Sci. 1999; 20: 157-161Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 47Takeo S. Nasa Y. Cardiovasc. Res. 1999; 43: 32-43Crossref PubMed Scopus (34) Google Scholar, 48Laclau M.N. Boudina S. Thambo J.B. Tariosse L. Gouverneur G. Bonoron-Adele S. Saks V.A. Garlid K.D. Dos Santos P. J. Mol. Cell. Cardiol. 2001; 33: 947-956Abstract Full Text PDF PubMed Scopus (93) Google Scholar), it is logical to predict that a similar mechanism will operate in brain (14Domoki F. Perciaccante J.V. Veltkamp R. Bari F. Busija D.W. Stroke. 1999; 30 (; discussion 2718–2719): 2713-2718Crossref PubMed Scopus (103) Google Scholar). This hypothesis can be more readily explored now that brain mitoKATP has been identified and its regulation partially characterized." @default.
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