ATP-sensitive Potassium Channel in Mitochondria of the Eukaryotic Microorganism Acanthamoeba castellanii
2007; Elsevier BV; Volume: 282; Issue: 24 Linguagem: Inglês
10.1074/jbc.m701496200
ISSN1083-351X
AutoresAnna Kicińska, Aleksandra Świda-Barteczka, Piotr Bednarczyk, Izabela Koszela-Piotrowska, Katarzyna Choma, Krzysztof Dołowy, Adam Szewczyk, Wiesława Jarmuszkiewicz,
Tópico(s)Ion channel regulation and function
ResumoWe describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP+-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mm ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 ± 7to166 ± 10 picosiemens, inhibition by 1 mm ATP/Mg2+ or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria. We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP+-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mm ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 ± 7to166 ± 10 picosiemens, inhibition by 1 mm ATP/Mg2+ or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria. The mitochondrial inner membrane was originally thought to have a low permeability to ions. This was based on the assumption that ion flux would lead to energy dissipation and depolarization of the mitochondrial membrane potential (ΔΨ). However, there is now clear evidence that anion, monovalent, and divalent cation channels exist in the inner membrane of mitochondria (1Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1327) Google Scholar, 2O'Rourke B. Annu. Rev. Physiol. 2007; 69: 23.1-23.31Google Scholar). These channels have profound effects on mitochondrial metabolism and the efficiency of oxidative phosphorylation. Furthermore, it is now known that the opening of these channels can have either detrimental (leading to apoptosis or necrosis) or beneficial effects on the cell (protection against ischemic damage, apoptosis, oxidative stress (for review see Refs. 3Tsujimoto Y. Nakagawa T. Shimizu S. Biochim. Biophys. Acta. 2006; 1757: 1297-1300Crossref PubMed Scopus (148) Google Scholar and 4Ardehali H. O'Rourke B. J. Mol. Cell. Cardiol. 2005; 39: 7-16Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar).Potassium channels were first described in the inner membrane of liver mitochondria (5Inoue I. Nagase H. Kishi K. Higuti T. Nature. 1991; 352: 244-247Crossref PubMed Scopus (666) Google Scholar). Later, similar mitochondrial ATP-sensitive potassium (mitoKATP) 2The abbreviations used are: mitoKATP channel, mitochondrial ATP-sensitive potassium channel; BLM, black lipid membrane; BSA, bovine serum albumin; CATR, carboxyatractylozide; 5-HD, 5-hydroxydecanoic acid; KCO, potassium channel opener; pS, picosiemens; SMP, submitochondrial particles; TPP+, tetraphenylphosphonium. 2The abbreviations used are: mitoKATP channel, mitochondrial ATP-sensitive potassium channel; BLM, black lipid membrane; BSA, bovine serum albumin; CATR, carboxyatractylozide; 5-HD, 5-hydroxydecanoic acid; KCO, potassium channel opener; pS, picosiemens; SMP, submitochondrial particles; TPP+, tetraphenylphosphonium. channels were described in other mammalian mitochondria, i.e. heart (6Paucek 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), brain (7Bajgar R. Seetharaman S. Kowaltowski A.J. Garlid K.D. Paucek P. J. Biol. Chem. 2001; 276: 33369-33374Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 8Debska G. May R. Kicinska A. Szewczyk A. Elger C.E. Kunz W.S. Brain Res. 2001; 892: 42-50Crossref PubMed Scopus (84) Google Scholar), skeletal muscle (9Debska G. Kicinska A. Skalska J. Szewczyk A. May R. Elger C.E. Kunz W.S. Biochim. Biophys. Acta. 2002; 1556: 97-105Crossref PubMed Scopus (89) Google Scholar), kidney (10Cancherini D.V. Trabuco L.G. Reboucas N.A. Kowaltowski A.J. Am. J. Physiol. 2003; 285: F1291-F1296Crossref PubMed Scopus (62) Google Scholar), and human T-lymphocytes (11Dahlem Y.A. Horn T.F. Buntinas L. Gonoi T. Wolf G. Siemen D. Biochim. Biophys. Acta. 2004; 1656: 46-56Crossref PubMed Scopus (75) Google Scholar) and also in plant mitochondria (12Pastore D. Stoppelli M.C. Di Fonzo N. Passarella S. J. Biol. Chem. 1999; 274: 26683-26690Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 13Ruy F. Vercesi A.E. Andrade P.B. Bianconi M.L. Chaimovich H. Kowaltowski A.J. J. Bioenerg. Biomembr. 2004; 36: 195-202Crossref PubMed Scopus (31) Google Scholar).Similar to the plasma membrane ATP-sensitive potassium channels (KATP channels), the mitoKATP channel is blocked by antidiabetic sulfonylureas and activated by potassium channel openers (KCOs) (14Szewczyk A. Skalska J. Glab M. Kulawiak B. Malinska D. Koszela-Piotrowska I. Kunz W.S. Biochim. Biophys. Acta. 2006; 1757: 715-720Crossref PubMed Scopus (71) Google Scholar, 15Mannhold R. Curr. Top. Med. Chem. 2006; 6: 1031-1047Crossref PubMed Scopus (25) Google Scholar). Diazoxide is an especially potent activator of the mitoKATP channel (16Garlid 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 (386) Google Scholar). The molecular identity of the mitoKATP channel is unknown. Several observations on the pharmacological profile and immunoreactivity with specific antibodies suggest that the mitoKATP channel belongs to the inward rectifier K+ channel family Kir6.x (17Suzuki M. Kotake K. Fujikura K. Inagaki N. Suzuki T. Gonoi T. Seino S. Takata K. Biochem. Biophys. Res. Commun. 1997; 241: 693-697Crossref PubMed Scopus (142) Google Scholar, 18Zhou M. Tanaka O. Sekiguchi M. Sakabe K. Anzai M. Izumida I. Inoue T. Kawahara K. Abe H. Brain Res. 1999; 74: 15-25Crossref Scopus (74) Google Scholar). Recently, it has been hypothesized that succinate dehydrogenase forms part of a structure that constitutes the mitoKATP channel (19Ardehali H. Chen Z. Ko Y. Mejia-Alvarez R. Marban E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11880-11885Crossref PubMed Scopus (187) Google Scholar).The primary function of the mitoKATP channel is to allow K+ transport into the mitochondrial matrix. This can lead to an increase in matrix volume and matrix alkalinization as well as an increase in reactive oxygen species production by mitochondria (for review see Ref. 20Garlid K.D. Paucek P. Biochim. Biophys. Acta. 2003; 1606: 23-41Crossref PubMed Scopus (301) Google Scholar). Additionally, in mammalian cells, it is now well established that mitoKATP channels play an important role in protecting cells against ischemia-reperfusion-induced injury. It has been found that an increase in the inner mitochondrial membrane permeability to K+ ions improves cellular tolerance to ischemia-reperfusion injury in various tissues, including liver, gut, brain, kidney, and heart (for review see Ref. 2O'Rourke B. Annu. Rev. Physiol. 2007; 69: 23.1-23.31Google Scholar).Although we now have quite a breadth of knowledge about ion transport in mammalian mitochondria, thus far nothing has been elucidated about cation-transporting systems in the mitochondria of unicellular eukaryotes. Acanthamoeba castellanii, a non-photosynthesizing amoeboid protozoon, is an especially interesting example of eukaryotic microorganisms, as in molecular phylogenesis appears on a branch basal to the divergence points of the animal, plant, and fungal kingdoms (21Grey M.W. Burger G. Lang B.F. Science. 1999; 283: 1476-1481Crossref PubMed Scopus (1347) Google Scholar). A. castellanii and higher plants share several common features at the level of the respiratory chain of the inner mitochondrial membrane, such as the presence of an alternative cyanide-resistant ubiquinol oxidase and nonphosphorylating rotenoneinsensitive internal (matrix face) and external (cytosolic face) NADH dehydrogenases (22Jarmuszkiewicz W. Wagner A.M. Wagner M.J. Hryniewiecka L. FEBS Lett. 1997; 11: 110-114Crossref Scopus (60) Google Scholar, 23Jarmuszkiewicz W. Sluse-Goffart C.M. Hryniewiecka L. Michejda J. Sluse F.E. J. Biol. Chem. 1998; 273: 10174-10180Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Mackenzie S. McIntosh L. Plant Cell. 1999; 11: 571-585Crossref PubMed Scopus (270) Google Scholar). On the contrary, recent studies on the translocase of the outer membrane (TOM) complex of A. castellanii mitochondria suggest that it could be rather similar to the animal-fungal TOM proteins (25Wojtkowska M. Szczech N. Stobienia O. Jarmuszkiewicz W. Budzinska M. Kmita H. J. Bioenerg. Biomembr. 2005; 37: 261-268Crossref PubMed Scopus (11) Google Scholar). As mitochondrial potassium channels are present in higher plant mitochondria and animal mitochondria, it is of special interest to explore its existence in ameboid protozoon. Identification and characterization of the mitoKATP channel in A. castellanii mitochondria, the subject of the present study, indicate that the K+ transporting system emerged early during phylogenesis, prior to the divergence of eukaryotes into animals and plants.EXPERIMENTAL PROCEDURESChemicals—l-α-Phosphatidylcholine (asolectin), diazoxide, glibenclamide, and n-decane were from Sigma-Aldrich. All other chemicals were of the highest purity available commercially.Cell Culture and Isolation of Mitochondria—The soil amoeba A. castellanii, strain Neff, was cultured as described by Jarmuszkiewicz et al. (22Jarmuszkiewicz W. Wagner A.M. Wagner M.J. Hryniewiecka L. FEBS Lett. 1997; 11: 110-114Crossref Scopus (60) Google Scholar). Trophozoites of the amoeba were collected between 44 and 48 h following inoculation at the middle exponential phase (at a density of about 4–6 × 106 cells/ml). Mitochondria were isolated and purified on a self-generating Percoll gradient (30%) as described earlier (21Grey M.W. Burger G. Lang B.F. Science. 1999; 283: 1476-1481Crossref PubMed Scopus (1347) Google Scholar). Mitochondrial protein concentration was determined by the biuret method.Preparation of Submitochondrial Particles—Freshly prepared mitochondria were diluted to about 8 mg of protein/ml with 0.25 m sucrose and 20 mm Tris-HCl, pH 7.4, and subsequently frozen at –20 °C. After thawing, the suspension was sonicated five times for 30 s and centrifuged at 12,000 × g for 15 min to pellet the unbroken mitochondria. The supernatant was centrifuged at 34,000 × g (30 min) to pellet submitochondrial particles (SMP), fraction 1 (smaller particles). The supernatant was again centrifuged at 200,000 × g (2 h) to collect fraction 2 of SMP (larger particles). Fractions of SMP, which are the mitochondrial inner membrane-enriched fractions, were resuspended in 0.3 m sucrose and 10 mm Hepes-KOH, pH 7.2, at a concentration of 5 mg of protein/ml.Black Lipid Membrane (BLM) Measurements—Experiments were performed by using the black lipid membrane technique as described previously (26Bednarczyk P. Kicinska A. Kominkova V. Ondrias K. Dolowy K. Szewczyk A. J. Membr. Biol. 2004; 199: 63-72Crossref PubMed Scopus (56) Google Scholar, 27Hordejuk R. Lobanov N.A. Kicinska A. Szewczyk A. Dolowy K. Mol. Membr. Biol. 2004; 21: 307-313Crossref PubMed Scopus (11) Google Scholar). In brief, BLMs were formed in a 250-μm-diameter hole drilled in a Delrin cup (Warner Instrument Corp., Hamden, CT), which separated two chambers (cis and trans, each 1 ml internal volume). The chambers contained 50/450 mm KCl (cis/trans) and 20 mm Tris-HCl, pH 7.2. The outline of the aperture was coated with a lipid solution and N2 dried prior to bilayer formation to improve membrane stability. BLMs were painted using asolectin in n-decane at a final concentration of 25 mg of lipid/ml. Amoeba A. castellanii SMP (about 5 μg of protein/ml, 0.5–1.5 μl/reconstitution) were added to the trans compartment (Fig. 4C). Incorporation of the potassium channel into the BLM was usually observed within a few minutes. The studied compounds were added to the cis and trans compartments. All measurements were carried out at 24 °C. The formation and thinning of the bilayer were monitored by capacitance measurements and optical observations. Final accepted capacitance values ranged from 110 to 180 picofarads. Electrical connections were made by Ag/AgCl electrodes and agar salt bridges (3 m KCl) to minimize liquid junction potentials. Voltage was applied to the cis compartment of the chamber, and the trans compartment was grounded (Fig. 4C). The current was measured using a bilayer membrane amplifier (BLM-120, BioLogic). Signals were filtered at 500 Hz. The current was digitized at a sampling rate of 100 kHz (A/D converter PowerLab 2/20, ADInstruments) and transferred to a PC for off-line analysis by Chart version 5.2 (PowerLab, ADInstruments) and pCLAMP 8.1 (Axon Laboratory). The pCLAMP 8.1 software package was used for data processing. The channel recordings presented are representative of the most frequently observed conductance values under the given conditions. The conductance was calculated from the currentvoltage relationship. The permeability ratios for K+ and Cl– were calculated according to the Goldman-Hodgkin-Katz voltage equation (28Hille B. Selective Permeability: Independence. Sinauer Associates Inc., Sunderland, MA2001: 441-470Google Scholar), Erev=-RTzFIn[Cl]cis+(PK/PCl)[K]trans[Cl]trans+(PK/PCl)[K]cis(Eq. 1) where Erev is the potential at which the current is zero, R is the gas constant, T is temperature in Kelvin, z is equal to –1 (chloride anion charge), F is Faraday constant, Pion is the permeability of the ion, and [Cl] and [K] are the respective concentrations of the ion in the cis and trans chambers.Mitochondrial Oxygen Consumption—Oxygen uptake was measured polarographically with a Clark-type oxygen electrode (Rank Brothers, Cambridge, United Kingdom) in 3 ml of incubation medium (25 °C), as described in a given figure legend (Figs. 1, 2, 3), with 1–2 mg of mitochondrial protein. Benzohydroxymate was used to inhibit the alternative oxidase activity. Succinate (5 mm) plus rotenone (4 μm), malate (5 mm), or NADH (1 mm) was used as the respiratory substrate. Measurements of the respiratory rate were performed in the absence of added ADP, i.e. in the resting state (state 4). To exclude the activity of an ATP/ADP antiporter, 1.8 μm carboxyatractylozide (CATR) was used. State 3 (phosphorylating) respiration measurements were performed (in the high KCl medium with 0.65 MgCl2 (23Jarmuszkiewicz W. Sluse-Goffart C.M. Hryniewiecka L. Michejda J. Sluse F.E. J. Biol. Chem. 1998; 273: 10174-10180Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar)) to check the coupling parameters. Only high quality mitochondria preparations, i.e. with an ADP/O value of around 1.40 and a respiratory control ratio of around 3 (with succinate), were used in all experiments. Values of O2 uptake are presented in nmol of oxygen × min–1 × mg–1 protein.FIGURE 1Influence of potassium channel activity modulating substances (diazoxide, glibenclamide, BMS-191095, and 5-HD) on resting (state 4) respiratory rate and membrane potential (ΔΨ) in isolated A. castellanii mitochondria. Mitochondria were incubated in the presence of 5 mm succinate (SA) and 4 μm rotenone in medium containing 20 mm Tris-HCl, 120 mm KCl, 0.5 mm MgCl2, 3 mm KH2PO4, 1.5 mm EGTA, 1.8 μm CATR, 0.2% BSA, and 5 mm benzohydroxamate. 100 μm diazoxide (diaz), 10 μm glibenclamide (glib), 3 μm BMS-191095, or 500μm 5-HD was added as indicated. Examples of nine (A) or six (B and C) measurements (using mitochondria from three different preparations) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2The effect of diazoxide concentration on resting respiratory rate and membrane potential (ΔΨ). Mitochondria were incubated in medium containing 20 mm Tris-HCl, 120 mm KCl, 0.5 mm MgCl2, 3 mm KH2PO4, 1.5 mm EGTA, 1.8 μm CATR, 0.2% BSA, and 5 mm benzohydroxamate (▪, ▴) or 20 mm Tris-HCl, 120 mm NaCl, 0.5 mm MgCl2, 3 mm NaH2PO4, 1.5 mm EGTA, 0.2% BSA, and 5 mm benzohydroxamate (▾). A and B, with 5 mm succinate (SA) (plus 4 μm rotenone) as a respiratory substrate. C and D, with 5 mm malate (MA) as a respiratory substrate. Under certain conditions ▴, 10μm glibenclamide (glib) was applied additionally. The data deal with three different mitochondrial preparations. S.D. is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Influence of potassium channel activity modulating substances (diazoxide and ATP) on resting respiratory rate and membrane potential (ΔΨ). Mitochondria were incubated in the presence of 1 mm NADH (as a respiratory substrate) and 4 μm rotenone in the medium described in the legend to Fig. 1. A and B, 100 μm diazoxide (diaz) and 1 mm ATP were added as indicated. Examples of nine measurements (using mitochondria from three different preparations) are shown. C, effect of diazoxide concentration on ΔΨ in the presence or absence of ATP. ▴, 1 mm ATP was added to the incubation medium before diazoxide addition after mitochondria. The data deal with three different mitochondrial preparations. S.D. is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mitochondrial Membrane Potential Measurements—The mitochondrial transmembrane electrical potential (ΔΨ) was measured simultaneously with oxygen uptake using a tetraphenylphosphonium (TPP+)-specific electrode according to Kamo et al. (29Kamo N. Muratsugu M. Hongoh R. Kobatake Y. J. Membr. Biol. 1979; 49: 105-121Crossref PubMed Scopus (886) Google Scholar). Measurements were performed in the presence of 1.3 μm TPP+. For calculation of the ΔΨ value, the matrix volume of amoeba mitochondria was assumed to be 2.0 μl × mg–1 protein. Calculation assumes that TPP+ distribution between mitochondria and medium followed the Nernst equation. Corrections were made for the binding of TPP+ to mitochondrial membranes. Values of ΔΨ are presented in mV.SDS-PAGE and Immunoblotting—Protein content was determined by the Bradford method (Bio-Rad). Samples (150 μg) of isolated mitochondrial proteins or SMP were solubilized in sample buffer containing 2% (w/v) SDS, 50 mm Tris/Cl, pH 6.8, 10% glycerol, 0.004% (w/v) bromphenol blue, and 8% mercaptoethanol) and subsequently were boiled for 4 min. Proteins were separated on 12% SDS-polyacrylamide gels and, after separation, electrotransferred to a nitrocellulose membrane. Membranes were then hybridized with anti-Kir6.1 and anti-Kir6.2 antibodies (at dilutions of 1:100) (Santa Cruz Biotechnology) in the presence or absence of blocking peptide. Protein bands were visualized using the GE Healthcare ECL system.RESULTSActivation of K+ Flux through the A. castellanii Mitochondrial Membrane by Potassium Channel Openers—The addition of a mitochondrial potassium channel opener, 100 μm diazoxide, increases the rate of mitochondrial state 4 oxygen uptake (with succinate) by 10.0 ± 1.6% (n = 9, S.D.). This effect is reversed to the control value by a potassium channel blocker, 10 μm glibenclamide. At the same time, ΔΨ decreases after addition of diazoxide by 1.2 ± 0.4 mV (n = 9, S.D.) and afterward is restored to the resting state value by glibenclamide. An example of simultaneously performed measurements of the resting respiratory rate and ΔΨ under the conditions described is shown in Fig. 1A. These results suggest that diazoxide stimulates the K+ ion flux into A. castellanii mitochondria, decreasing the steady state ΔΨ and thus causing the acceleration of mitochondrial respiration rate. To further test this hypothesis, we performed the same experiments in media deprived of K+ ions. In a sucrose (0.25 m) medium (data not shown) and in NaCl (0.12 m) medium (Fig. 2, A and B) the addition of either diazoxide or glibenclamide did not change the control values of resting respiration and ΔΨ in A. castellanii mitochondria. Another potassium channel opener, described previously as specific for the mitoKATP channel (30Grover G.J. D'Alonzo A.J. Garlid K.D. Bajgar R. Lodge N.J. Sleph P.G. Darbenzio R.B. Hess T.A. Smith M.A. Paucek P. Atwal K.S. J. Pharmacol. Exp. Ther. 2001; 297: 1184-1192PubMed Google Scholar), BMS-191095, also was applied in our studies. Similar to diazoxide, 3 μm BMS-191095 stimulated mitochondrial resting oxygen uptake by 8.9 ± 2.5% (n = 4, S.D.) and decreased ΔΨ by 1.2 ± 0.1 mV (n = 4, S.D.). Both effects were reversed by the addition of 10 μm glibenclamide. A representative experiment is shown in Fig. 1B.Fig. 1C shows that another previously described potassium channel blocker, 5-hydroxydecanoic acid (5-HD) (16Garlid 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 (386) Google Scholar), is not active when used with amoeba mitochondria. The addition of 500 μm 5-HD did not change the mitochondrial respiration rate and ΔΨ accelerated by diazoxide (Fig. 1C).Fig. 2, A and B, shows the diazoxide concentration-dependent effects on resting respiratory rate (stimulation) and ΔΨ (depolarization) with succinate-oxidizing A. castellanii mitochondria in the KCl-based medium. The apparent 50% maximal effect of diazoxide (K0.5) is reached at 43–45 μm. Concentrations of diazoxide higher than 100 μm were described to have nonspecific effects on mitochondria (31Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Am. J. Physiol. 2001; 280: H649-H657Crossref PubMed Google Scholar) and therefore have not been used in this study. Moreover, it is clear from Fig. 2, A and B, that the effects of diazoxide action are strictly dependent on the K+ ion presence in the incubation medium (no effects in a NaCl-based medium) and reversed by 10 μm glibenclamide. Furthermore, the dose-dependent and glibenclamide-blocked influence of diazoxide on mitochondrial bioenergetics is also observed during malate-sustained respiration (Fig. 2, C and D). In the case of malate, mitochondrial respiration is stimulated up to about 11%, whereas ΔΨ is maximally decreased by 1.5 mV. The apparent value of K0.5 is observed at 52–53 μm diazoxide.The inhibitory effect of ATP on K+ transport into A. castellanii mitochondria has been studied with external NADH as a respiratory substrate in order to exclude the influence of the nucleotide on malate and succinate dehydrogenase activities (23Jarmuszkiewicz W. Sluse-Goffart C.M. Hryniewiecka L. Michejda J. Sluse F.E. J. Biol. Chem. 1998; 273: 10174-10180Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). With mitochondria oxidizing external NADH, 100 μm diazoxide stimulates respiration by 15.7 ± 2.5% (n = 9, S.D.) and decreases ΔΨ by 1.30 ± 0.45 mV (n = 9, S.D.). The apparent value of K0.5 is estimated at around 50 μm diazoxide (Fig. 3C). The effect of diazoxide is abolished by the addition of 1 mm ATP (Fig. 3, A and C). Moreover, diazoxide was not able to activate potassium transport (no effect on either respiratory rate or ΔΨ was observed) when added after ATP (Fig. 3B). These results indicate that the nucleotide is an inhibitor of potassium transport in A. castellanii mitochondria. Moreover, in the K+ ion-deprived medium (0.25 m sucrose), the inhibitory effect of ATP was not observed (data not shown). The presence of 1.8 μm CATR in experiments where ATP was applied is especially important to exclude the adenine nucleotide translocator action and thereby ATP uptake into mitochondrial matrix. The same results were also observed in the presence of oligomycin (1 μg/mg of protein), a F1F0-ATP synthase inhibitor.The above functional characterization, drawn from experiments performed with isolated mitochondria, suggests that the potassium transport mechanism, similar to that observed in mitochondria of higher eukaryotes (plants and mammals), is present in A. castellanii mitochondria. To support these results, a further characterization of potassium transport with the inner mitochondrial membrane-enriched fractions (SMPs) reconstituted in a planar lipid bilayer was performed.Characterization of Potassium Channel from A. castellanii Mitochondrial Membrane in BLMs—The particular inner mitochondrial membrane-enriched fractions (SMP fractions 1 and 2) from the amoeba A. castellanii were reconstituted into BLMs as described under "Experimental Procedures." The results obtained upon reconstitution indicate no functional distinction between both fractions of particles. A variety of channel-like activities has been observed, both anion- and cation-selective (n = 117). Among them, we have focused on the K+ ion transport. Fig. 4A shows representative current-time traces and current-voltage relationship for potassium channel opening at different voltages in the 50/450 mm KCl (cis/trans) gradient solutions. The calculated channel conductance is equal to 166 ± 10 pS for potential from –30 to +30 mV and 90 ± 7 pS for potential from 50 to 110 mV. The reversal potential of 47 mV (Fig. 4B) has been calculated from measurements in the 50/450 mm KCl gradient solutions and curve fitting to the experimental data. This value indicates that the examined ion channel is a cation-selective. The calculated permeability ratio for K+ and Cl– is equal to 20.4 according the Goldman-Hodgkin-Katz voltage equation (28Hille B. Selective Permeability: Independence. Sinauer Associates Inc., Sunderland, MA2001: 441-470Google Scholar) (see Equation 1 under "Experimental Procedures").Substances known to modulate the mitoKATP channel activity have been used to examine the properties of the K+ ion channel observed in our experiments. Fig. 5A shows the single channel recordings in the 50/450 mm KCl (cis/trans) gradient solutions at 0 mV, before and after the addition of 1 mm ATP/Mg2+ to both chambers (cis/trans). ATP/Mg2+ causes a complete inhibition of the channel. The effect has usually been observed after about 5 min following ATP/Mg2+ addition. The result is clearly depicted in histograms shown below the current-time trace (Fig. 5A). The number of observations of the channel being in an open state changes from 61% to zero. The next recording (Fig. 5B) presents the inhibitory effect of 50 μm glibenclamide after its addition to the cis and trans compartments. Again, we observe the complete transition of the channel protein into a closed state, a few seconds after the inhibitor addition. The number of open state events changes from 50% to zero after glibenclamide addition, as illustrated in the histogram (Fig. 5B). The last trace on Fig. 5C shows that the inhibitory effect of ATP/Mg2+ is reversed by the addition of 30 μm diazoxide (cis/trans) in the presence of ATP/Mg2+ (cis/trans). The effect of diazoxide was usually evident within a few seconds after its application. Diazoxide causes the transition into an open state in 67% of events (histogram below the trace, Fig. 5C).FIGURE 5Influence of ATP/Mg2+, glibenclamide, and diazoxide on the activity of potassium channel from A. castellanii mitochondria. A, an example of the single channel recordings under control conditions and after addition of 1 mm Mg2+ and 1 mm ATP. B, an example of single channel recordings under control conditions and after addition of 50 μm glibenclamide. C, an example of single channel recordings in the presence of ATP/Mg2+ and after addition of 30 μm diazoxide. A–C, the amplitude histograms fitted with superimposed Gaussian curves are shown below the recordings. All experiments were performed in the 50/450 mm KCl (cis/trans) gradient solutions at 0 mV with at least three independent repetitions. All chemicals were added to cis and trans compartments. –, indicates the closed state of th
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