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

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 for the of the probe were monitored using an fluorescence with fluorescence to K+ flux as previously described K.D. S. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were as the values is the between in the and of 200 μm ATP, and is the between in the or of the mitoKATP and were from independent by to using DEAE-cellulose fractions containing mitoKATP in Triton were incubated for at 25 °C with 50 BODIPY-FL-glyburide, in the or of 1 μm were for at 4 °C G. F. U. S. Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and to probe PubMed Scopus Google Scholar). proteins were with in 50 mm pH 6.8, 20 times with 50 mm pH 6.8, and analyzed for fluorescence and BODIPY-FL-glyburide were from electrophoresis were from column and other were from We reconstituted brain mitoKATP using 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). A the active mitoKATP that was eluted from a DEAE-cellulose This contains protein and similar to those observed in active fractions from heart or liver mitochondria. purification of this on an ATP column a active containing and proteins 1 the proteoliposomes K+ flux of mitoKATP was for K+ flux a that the flux was K+ flux was inhibited by 200 μm ATP and this was by 50 μm cromakalim, a KATP previously observed with ATP not in the of (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 flux in liposomes reconstituted with brain mitoKATP. The contains representative of from fluorescence K+ into liposomes was initiated by the of 0.5 μm to provide K+ flux through mitoKATP was inhibited by 200 μm ATP and ATP was by 50 μm cromakalim a in which was contains the of to determine the of K+ flux on ATP and The K12 for ATP was 3 μm The K12 for opening in the of 200 μm ATP was MitoKATP was also from ATP by the KATP channel openers diazoxide and cromakalim The channel the of 200 μm ATP and 2 was inhibited by or previously not ATP, and diazoxide were (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). and other are in It be that mitoKATP were at similar to those found for heart and liver of mitoKATP heart and μm 0.2 μm μm μm μm μm are from rat brain mitoKATP with from heart and liver mitoKATP. The were from such as those contained in The were previously are in in a new of brain mitoKATP by diazoxide and The mitoKATP flux is of the KATP channel openers diazoxide and cromakalim in the of 200 are representative of of brain mitoKATP by and The mitoKATP flux is of the KATP channel blockers and 5-hydroxydecanoate K12 in the of 200 μm ATP and 2 μm are representative of are from rat brain mitoKATP with from heart and liver mitoKATP. The were from such as those contained in The were previously are in to K+ in brain mitochondria was 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 shown in There is an of K+ salts and which to the matrix K+ that was mitochondrial K.D. of Mitochondrial Mitochondrial Scholar). A is which a net flux between K+ and K+ the mitochondrial K.D. Biochim. Biophys. Acta. 1996; PubMed Scopus Google Scholar). was in and by μm ATP and the were by of 10 μm diazoxide was inhibited by further of 2 μm effects of ATP, diazoxide, or were observed or were for medium K+ not these are for K+ and to opening and closing of mitoKATP. The effects of mitoKATP on mitochondrial 2 in brain mitochondria are shown in Respiration was in K+ and We previously that opening of heart mitoKATP is with small in that to a K+ of A.J. S. Paucek P. Garlid K.D. J. Physiol. Circ. Physiol. PubMed Google Scholar). Brain mitochondria a significantly in respiration, to of an of 10 this to of seven times than that observed in rat heart mitochondria. this of K+ not the mitochondrial membrane using fluorescence Lett. PubMed Scopus Google Scholar) that by which is with the of the of brain mitoKATP, we were by the that of were to transport to heart and liver. mg of rat brain mitochondria similar to from 100 mg of rat liver or heart mitochondria. This was with the of K+ from We to the of an independent et al. G. F. U. S. Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar) shown that the sulfonylurea receptor be in and we the same to labeling of mitoKATP. The of these studies are contained in was seven times more milligram of mitochondrial in brain than in with the transport activity in brain mitochondria. In further studies for we the ATP column eluate with BODIPY-FL-glyburide μm and the proteins by SDS-PAGE. The protein was whereas the protein was not of mitoKATP in brain and liver protein fluorescence protein was in by BODIPY-FL-glyburide, as described in and the fluorescence was to protein in a new MitoKATP was in by BODIPY-FL-glyburide, as described in and the fluorescence was to protein We report 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 is by GTP, diazoxide, or cromakalim, and the channel is inhibited by or The to and the of two protein in the purified that mitoKATP is a of a inwardly rectifying K+ Google Scholar, Yarov-Yarovoy Biol. 1996; Scholar), and a sulfonylurea G.J. Garlid K.D. J. Mol. Cell. Cardiol. 2000; 32: 677-695Abstract Full Text PDF PubMed Scopus (384) Google Scholar). of mitoKATP in regulation of matrix is by the effects of ATP, diazoxide, and shown in effects are similar to those observed in heart mitochondria A.J. S. Paucek P. Garlid K.D. J. Physiol. Circ. Physiol. PubMed Google Scholar) and are to the regulation mediated by the mitochondrial K+ in as described in the to K+ due to mitoKATP opening a in which to a K+ flux of This of due to K+ is it is that it is times than that observed in heart or liver mitochondria A.J. S. Paucek P. Garlid K.D. J. Physiol. Circ. Physiol. PubMed Google Scholar), a that was by BODIPY-FL-glyburide labeling of the purified proteins The for the in brain is The of is 10 Biol. 1997; Google Scholar), which to a of of K+ per of channel protein per by the an of of channel per mg of brain mitochondrial are and per and mitoKATP is of the time we that brain mitochondria contain of and per mg of There is in the mechanism of protection against ischemia-reperfusion that heart and brain of ischemic and it is that KATP channels play an important both are protected by ischemic preconditioning in which a of against a of R.B. Circ. Res. PubMed Scopus Google Scholar, C. Widmann C. Lazdunski M. Proc. Natl. Acad. Sci. U. S. 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