Experimental Neurology 257 (2014) 70–75
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Hemin inhibits the large conductance potassium channel in brain mitochondria: A putative novel mechanism of neurodegeneration Bartłomiej Augustynek a,b, Alexei P. Kudin b, Piotr Bednarczyk a,c, Adam Szewczyk a,⁎, Wolfram S. Kunz b a b c
Laboratory of Intracellular Ion Channels, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland Department of Epileptology, University of Bonn, 25 Sigmund-Freud Street, D-53105 Bonn, Germany Department of Biophysics, Warsaw University of Life Sciences, 159 Nowoursynowska Street, 02-776 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 10 December 2013 Revised 14 April 2014 Accepted 23 April 2014 Available online 30 April 2014 Keywords: Mitochondria Hemin Intracerebral hemorrhage Mitochondrial BKCa channel Neurodegeneration ROS synthesis
a b s t r a c t Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca2+-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca2+-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca2+-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH. © 2014 Elsevier Inc. All rights reserved.
Introduction Intracerebral hemorrhage is a type of intracranial bleeding that occurs within the brain tissue. It can either be caused by brain trauma or occur in hemorrhagic stroke. Stroke is the second most common cause of death worldwide (Donnan et al., 2008), and hemorrhagic strokes account for 10–15% of all stroke cases (Sudlow and Warlow, 1997). Mortality of hemorrhagic stroke is high, and has not fallen within the last years. According to different studies it varies from 31% at 7 days to more than 90% at 10 years (Flaherty et al., 2006; Fogelholm et al., 2005). In hemorrhagic stroke the rapture or leak of a blood vessel leads to the formation of hematoma which oppresses the surrounding tissue and hampers their supply in oxygen and nutrients. Cells within and adjacent to the hematoma die quickly and there is not much that can be done today to prevent it. However, there is a pool of cells that experience negative effects of hemorrhage, although do not die immediately. Such cells would be a suitable target for potential therapies. It
⁎ Corresponding author. E-mail address: [email protected] (A. Szewczyk).
http://dx.doi.org/10.1016/j.expneurol.2014.04.022 0014-4886/© 2014 Elsevier Inc. All rights reserved.
is widely believed that one of the causes of cell loss in hemorrhagic stroke is the erythrocyte lysis that occurs in hematoma. Neurotoxic agents like hemoglobin, and its breakdown product hemin migrate from hematoma into surrounding tissue impairing the proper function of nervous cells (Chen-Roetling and Regan, 2006; Dang et al., 2011b). Hemin liberated from the hematoma can reach concentrations of as high as 10 mM (Robinson et al., 2009). Both astrocytes and neurons express heme carrier protein 1 (HCP1) in the plasma membrane, and hence can accumulate hemin upon its release from disintegrating erythrocytes (Dang et al., 2010, 2011b). Recent experimental data show rapid uptake of hemin by all brain resident cells, especially by microglia (Chen-Roetling et al., 2014). Additionally, hemin is a lipophilic compound that can intercalate into lipid bilayers, and cross them. However, one should expect that this phenomenon is rather of minor importance for hemin accumulation by cells. Still, it may play a role in hemin trafficking within the cell. Although neurotoxicity of hemin has been well documented, molecular basis of this phenomenon remains unclear. There are many processes and proteins which function can be impaired by this agent. One of them, the large-conductance Ca2 +-regulated potassium channel (BKCa channel) in the plasma membrane is inhibited by hemin (Tang et al., 2003).
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The BKCa channel is ubiquitously expressed in the plasma membrane of both excitable and non-excitable cells. It is activated by the changes in membrane potential and/or the calcium ions. Although it was extensively studied mainly in the plasma membrane, its counterparts exist within several intracellular structures like mitochondria (Siemen et al., 1999; Szabò et al., 2012). MitoBKCa is one of the best described channels present in the inner mitochondrial membrane. It can be blocked by charybdotoxin (ChTx), iberiotoxin (IbTx) or paxilline (Pax) and stimulated by calcium ions, changes in membrane potential, or by certain synthetic agents like NS1619. The large conductance calcium regulated potassium channel was identified in mitochondria of several cell types, namely: human glioma cell line LN229 (Siemen et al., 1999), guinea pig ventricular cells (Xu et al., 2002), skeletal muscle (Skalska et al., 2008), endothelial cells (Bednarczyk et al., 2013a), astrocytes (Thiede et al., 2012), astrocytoma (Bednarczyk et al., 2013b) and neurons (Fahanik-Babaei et al., 2011; Piwonska et al., 2008; Skalska et al., 2009). Although BKCa channels exist in many different locations throughout the cell, all of their forms share functional characteristics with their plasma membrane counterpart and all are encoded by a single gene: Kcnma1. Hence, it is widely believed that different types of BKCa channels are the results of alternative splicing of this gene. Although, the molecular identity of the mitoBKCa channel still remains to be elucidated, in 2013 Singh and co-workers have shown that a 50-aa C-terminal splice insert determines the mitochondrial location of mitoBKCa (Singh et al., 2013). Potassium channels are notorious for their role in cytoprotection (Escande and Cavero, 1992; Garlid, 2000; Malinska et al., 2010). Such cytoprotection could be especially important for tissues with a poor endogenous antioxidant defense such as the cardiac muscle or the nervous system where undisturbed and continuous blood flow and oxygen supply are extremely important (Doré, 2002). It is broadly accepted that preconditioning with the potassium channel openers (KCOs) results in cytoprotection throughout activation of mitochondrial channels (Facundo et al., 2006). The basis of KCO cytoprotective properties still remains to be elucidated, although it is suspected that attenuation of ROS synthesis in mitochondria that is observed after KCO administration may play some role in this phenomenon. Under substrate conditions that allow reverse electron flow, matrix K+ influx through the open mitoBKCa channel is believed to accelerate forward electron transfer and inhibit ROS production (Heinen et al., 2007a). Acceleration of ROS synthesis rate in turn is linked with several neurodegenerative diseases (Popa-Wagner et al., 2013). Another possible explanation of the cytoprotective properties of KCOs is mild uncoupling. The opening of mitochondrial potassium channels results in a modest dissipation of proton motive force by contributing inward current and following utilization of some of the proton gradient for K+ ejection from the matrix through the K+/H+ exchanger (O'Rourke, 2004) leading to decreased ROS synthesis (Kulawiak et al., 2008). The goal of this study was to elucidate whether hemin can inhibit the large conductance calcium regulated potassium channel in the mitochondrial inner membrane and if so, how does it alter some of the known beneficial effects of potassium channel openers on cell function.
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Cell culture and preparation of astrocytoma mitochondria Mitoplasts for patch-clamp experiments were prepared from the human astrocytoma U-87 MG cell line. The cell line identity was confirmed by the short tandem repeat (STR) profiling technique. This assay was performed according to the guidelines published by Masters et al. (2001). More details are in Bednarczyk et al. (2013b). For the experiments, cells were cultured in DMEM medium (10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin) at 37 °C in a humidified atmosphere with 5% CO2. The cells were fed and reseeded every third day. Once reached a confluence of about 70%, cells from five 75 cm2 flasks were scraped and centrifuged at 800 ×g for 10 min, resuspended in the preparation solution for the mitochondrial isolation (250 mM sucrose, 5 mM HEPES, pH = 7.2) and homogenized. First (9200 ×g, 10 min) and second (770 ×g, 10 min) centrifugation steps were performed to separate the fraction enriched in mitochondria. Sucrose was removed by two further fast centrifugation steps (9200 ×g, 10 min) in the storage solution (150 mM KCl, 10 mM HEPES, pH = 7.2). All procedures were performed at 4 °C. Patch clamp experiments Patch-clamp experiments on mitoplasts (spherical vesicles surrounded by the mitochondrial inner membrane) were performed as described previously (Bednarczyk et al., 2010, 2013b; Cheng et al., 2008; Toczyłowska-Mamińska et al., 2013). Briefly, a sample of purified human astrocytoma mitochondria was put into a hypotonic solution (5 mM HEPES, 200 μM CaCl2, pH = 7.2) for about 1 min in order to induce swelling and disruption of the mitochondrial outer membrane. Then, the addition of a hypertonic solution (750 mM KCl, 30 mM HEPES, 200 μM CaCl2, pH = 7.2) restored the isotonicity of the medium, and stopped further swelling. Mitoplasts were distinguished from the cellular debris present in preparation by their transparency, spherical shape, and characteristic ‘cap’ — remain of the outer membrane. In each experiment, one free-floating mitoplast was attached to the patch-clamp pipette made of borosilicate glass filled with an isotonic solution (150 mM KCl, 10 mM HEPES, 200 μM CaCl2, pH = 7.2). The isotonic solution was also used in control recordings for all presented data. Hemin and iberiotoxin (300 nM hemin and 2 nM IbTx in isotonic solution) were added from the back of the patch-clamp pipette through the peristaltic pump-driven capillary-pipe system. The experiments were carried out in patch-clamp inside-out mode. Reported voltages are those applied to the patch clamp pipette interior. The current was recorded by the patch-clamp amplifier Axopatch 200B (Molecular Devices Corporation). The pipettes had a resistance of 10–20 MΩ and were pulled by a Flaming/Brown type P-100 puller (Sutter Instrument). The measured ion currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. All experimental traces were recorded at the single-channel mode. The conductance was calculated from the current–voltage characteristics (data not shown). The probability of channel opening was determined using the singlechannel search mode of the Axon™ pCLAMP® 10 Electrophysiology Data Acquisition & Analysis Software (Molecular Devices Corporation).
Materials and methods
Isolation of rat brain mitochondria
All standard chemicals along with 1,3-dihydro-1-[2-hydroxy5(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), iberiotoxin (IbTx), charybdotoxin (ChTx), horseradish peroxidase (HRP) and hemin were purchased from Sigma-Aldrich. The uncoupler TTFB (4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole) was a kind gift from Prof. B. Beechey (Aberystwyth, UK). Catalase, digitonin and superoxide dismutase (SOD) were purchased from Serva, and bacterial proteinase from Fluka. All cell culture materials were purchased from Gibco.
Solutions used for: mitochondrial isolation: MSE solution (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES, 1 mg/ml essential fatty acid free BSA, pH = 7.4); measurements: K+ containing MTP medium (10 mM KH2PO4, 60 mM KCl, 60 mM Tris–HCl, 110 mM mannitol, 5 mM MgCl2, 0.5 mM EDTA, pH = 7.4) and K+ free MTP medium (10 mM NaH2PO4, 60 mM NaCl, 60 mM Tris–HCl, 110 mM mannitol, 5 mM MgCl2, 0.5 mM EDTA, pH = 7.4). Rat brain mitochondria were isolated according to the standard protocol described by Rosenthal et al. (1987), with a small modification
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that allowed obtaining mitochondria with much better functional characteristics (Kudin et al., 2004).
significance of the obtained results. P b 0.05 was considered statistically significant. Significance levels are: 0.05–0.01 (*), 0.01–0.001 (**), and b0.001 (***).
Measurements of H2O2 generation by rat brain mitochondria Results In the presence of HRP H2O2 oxidizes colorless p-hydroxyphenylacetic acid (pHPPA, 200 μM). This reaction results in the formation of its fluorescent dimer (pHPPA)2 (λex = 317 nm, λem = 414 nm). All measurements were performed with a RF-5001 spectrofluorophotometer (Shimadzu). Shortly, mitochondrial H2O2 generation was fueled by succinate (10 mM) and measured at 30 °C in an oxygen-saturated MTP medium. The reactions were catalyzed by HRP (20 U/ml) in the presence of mitochondria (0.2 mg of protein/ml). An excess of SOD (176 U/ml) guaranteed the complete conversion of all produced superoxide into hydrogen peroxide (Kudin et al., 2004; Malinska et al., 2009). The fluorescence signal was calibrated by a series of hydrogen peroxide additions (normally 6 additions of 140 pmol H2O2) in the presence of pHPPA and isolated mitochondria. Measurements of rat brain mitochondrial membrane potential Changes in the potential of the mitochondrial inner membrane (ΔΨm) were evaluated by the rhodamine 123 (2.6 μM) fluorescence quenching (λex = 450 nm, λem = 550 nm), measured with a RF-5001 spectrofluorophotometer (Shimadzu). The measurements were performed in MTP media at 30 °C. 10 mM glutamate and 5 mM malate were used to energize mitochondria. The concentration of rat brain mitochondrial protein was about 0.2 mg/ml. Addition of mitochondria causes a sharp drop in the rhodamine fluorescence. Minimal fluorescence corresponds to the maximal membrane potential. Any increase of the rhodamine fluorescence means dissipation of the ΔΨm. To calculate the mitochondrial membrane potential, the fluorescence changes were calibrated with corresponding potassium diffusion potentials. Briefly, 100 μM valinomycin was added to the rotenone de-energized mitochondrial preparation and incubated in a medium in which K+ ions were replaced by Na+. Next, the preparation was titrated with precisely defined additions of KCl until no further changes of rhodamine 123 fluorescence were recorded (equilibrium point). The corresponding potentials were calculated using the Nernst equation. Data analysis All experimental values are reported as mean ± SD (standard deviation). The Student's t test was used to determine the statistical
Patch-clamp Patch-clamp experiments were performed on mitoplasts obtained from the human astrocytoma U-87 MG cell line. All currents were measured in symmetric 150/150 mM KCl isotonic solution with 200 μM CaCl2. To determine whether the observed channel activity was associated with the BKCa we reduced the Ca2 + concentration from high (200 μM) to low (1 μM) and tested its sensitivity to the BKCa inhibitors. All low calcium conditions, iberiotoxin and paxilline inhibited channel activity (Bednarczyk et al., 2013b). Furthermore, at the end of each experiment, an addition of iberiotoxin (100 nM) was used to confirm the identity of the channel (immediate channel inhibition). Fig. 1A shows the single-channel recordings of mitoBKCa activity at different voltages in control conditions and after addition of 300 nM hemin. In Fig. 1B we present results from 9 experiments of such type in the context of channel activity, each comprising 1 min of recordings. This analysis revealed that hemin inhibits the activity of the channel by about 40% at +40 mV, and this inhibition is statistically significant. H2O2 synthesis in rat brain mitochondria In the current study we have measured succinate fueled H2O2 generation rate in the conditions that allow reverse electron flow. As it was previously shown, in these conditions addition of BKCa channel openers, such as NS1619, causes substantial attenuation of H2O2 generation rate (Kulawiak et al., 2008). According to our measurements, NS1619 (2.5 μM) attenuates brain mitochondrial ROS generation rate by reverse and forward electron flow by about 45%. As visible in Fig. 2A the addition of hemin (2.5 μM) inhibits this effect by about 30%. It is noteworthy, that the strength of this inhibition is comparable with that caused by iberiotoxin (5 μM) (Fig. 2B). Each experiment was finalized by an addition of catalase to inhibit further H2O2 generation. As one can see, the effectiveness of hemin/iberiotoxin attenuation of NS1619 effect was limited. It can be explained by the fact that a decrease in H2O2 synthesis rate caused by NS1619 comes from a combination of its specific properties towards the mitoBKCa channel and some unspecific actions. Therefore, hemin along with iberiotoxin abolishes probably only a part of the observed effect that results from inhibition of the mitoBKCa channel.
Fig. 1. Single channel recordings of the mitoBKCa channel in the presence of 300 nM hemin. A. Raw experimental traces (symmetric 150/150 mM KCl isotonic solution with 200 μM Ca2+) at different voltages, ‘–’ indicates closed state. B. Average probability of opening of the mitoBKCa channel upon addition of 300 nM hemin, n = 9.
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Fig. 2. ROS synthesis rate. A. Representative experimental traces showing the effect of: 2.5 μM NS1619, 2.5 μM NS1619 + 2.5 μM hemin and 2.5 μM NS1619 + 5 μM iberiotoxin on the H2O2 synthesis rate by isolated rat brain mitochondria (10 mM succinate, 200 μM pHPPA, 20 U/ml HRP, 176 U/ml SOD). AF — arbitrary fluorescence units. B. Average H2O2 synthesis rate after administration of 2.5 μM NS1619, 2.5 μM NS1619 + 2.5 μM hemin and 2.5 μM NS1619 + 5 μM iberiotoxin, n = 7.
The remaining (hemin/iberiotoxin-insensitive) NS1619 effect is likely caused by the unspecific properties of the opener (Debska et al., 2003; Heinen et al., 2007b; Kicinska and Szewczyk, 2004). Dissipation of mitochondrial inner membrane potential To investigate the possible impact of hemin on mitochondrial membrane potential throughout activation of the mitoBKCa channel we have performed a set of experiments employing fluorescent properties of rhodamine 123. The samples of rat brain mitochondria oxidizing glutamate and malate (10 mM, 5 mM respectively) were treated with NS1619 (2.5 μM). This resulted in a mild membrane potential dissipation (about 12 mV) (Fig. 3A). In the presence of 2.5 μM hemin (Fig. 3B), and 2.5 μM charybdotoxin (Fig. 3C), the effect of NS1619 on
membrane potential was substantially attenuated. Each experiment was completed by an addition of the uncoupler (TTFB, 1 μM) to deenergize the mitochondria. To prove that the observed effect is specific to potassium ion permeability, we have performed control experiments in a potassium free medium. In the absence of potassium ions, the reported influence of NS1619 on the membrane potential was comparable with the one observed after ChTx addition (Fig. 3D). We have also verified the relationship between concentration of hemin and its inhibitory effect on the opening of the mitoBKCa channel (Fig. 4). Hemin clearly acts in a dose-dependant manner. However, in these conditions the increase of dose above 3 μM does not significantly alter membrane potential any longer. Taken together, hemin can abolish about 50% of the NS1619 potential-dissipating properties.
Fig. 3. Changes in the membrane potential. A, B, C, D — Representative experimental traces for isolated brain mitochondria upon addition of 2.5 μM NS1619. The fluorescence of rhodamine 123 is inversely proportional to the membrane potential. mito — addition of isolated brain mitochondria; TTFB — uncoupler (1 μM), and AF — arbitrary fluorescence units.
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Fig. 4. Hemin dose response. Dissipation of the mitochondrial inner membrane potential upon addition of 2.5 μM NS1619 in the presence of increasing concentrations of hemin (1–4 μM), n = 12.
Discussion In 2003, Tang and co-workers have shown that heme and the product of its oxidation hemin can bind to, and inhibit the BKCa channel in the plasma membrane (Tang et al., 2003). Since an increase in the potassium ion permeability of the mitochondrial inner membrane contributes to cytoprotection (O'Rourke, 2004; Xu et al., 2002), it is possible that the phenomenon of mitoBKCa channel inhibition by hemin could exert some negative impact on cells. This effect would be particularly pronounced in the case of massive hemin influx which occurs in neurological conditions like intracerebral and subarachnoid hemolysis, hemorrhagic stroke or brain trauma. As it was previously mentioned, brain hemorrhages are potent sources of hemin that infiltrates to the interstitial space within brain tissue, and literally floods neurons, astrocytes and other brain cells. The issue of hemin toxicity in hemorrhagic stroke was comprehensively reviewed by Robinson et al. (2009). According to this group, neurons accumulate hemin 50% faster than astrocytes, and therefore are more susceptible to its cytotoxic properties. Moreover, they suggested that the extent of hemin toxicity cannot be simply explained by the mechanisms described so far (Dang et al., 2011a,b) and concluded that the molecular basis of hemin neurotoxicity appears to be multilevel, thus requires more attention. It has been previously proposed that the BKCa channel posses a highly conserved heme binding motive, the same as described in cytochrome c (Jaggar et al., 2005; Tang et al., 2003). According to the mechanism postulated by Jaggar and co-workers reduced heme is a physiological inhibitor of BKCa channels. It has the ability to bind carbon monoxide (CO) a molecule that is constantly produced in very small quantities by heme oxygenases. According to this model, heme can be considered as a cellular CO sensor. Once CO binds to the heme that is attached to a BKCa channel molecule it alters heme–channel interaction that results in its activation. In light of these reports inhibitory effect of hemin can rely on the replacing of the reduced heme molecules by oxidized hemin. Hemin cannot bind CO; hence it maintains the channel in the closed state. This model seems to be even more valid when we consider data published by Yi and co-workers in 2010. According to them, the BKCa channel can directly interact with heme oxygenase-2 (a CO producer). They have described a sophisticated thiol/disulfide switch in the heme binding domain of the channel, claiming that this is the mechanism by which the activity of the BKCa channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions (Yi et al., 2010). Summarizing, the strong evidences prove that the BKCa channel along with heme oxygenase, heme and CO can take part in a sophisticated mechanism that allow the cell to detect changes in its internal redox state. It is possible that similar mechanisms regulate the mitoBKCa channel. Mitochondria are the cellular power plants relying on continuous redox reactions that drive the ATP synthesis. Therefore, it seems
likely that a redox state-sensitive ion channel (e.g. mitoBKCa) could play an important role in the proper function of mitochondria. The results presented in the current work provided to our knowledge the first proof that hemin can inhibit the mitoBKCa channel. This in turn diminishes the beneficial effects of mitoBKCa channel opening such as dissipation of the membrane potential and the concomitant decrease in ROS synthesis rate. According to recently published work (Bednarczyk et al., 2013b), there is a putative structural and functional coupling of the mitoBKCa channel with the elements of the mitochondrial respiratory chain in the human astrocytoma U-87 MG cell line. It appeared that acceleration of the electron transfer by addition of its substrates inhibits activity of the mitoBKCa channel and this effect can be abolished by the inhibitors of the respiratory chain. It was showed that there is a putative interaction between the regulatory subunit of the mitoBKCa channel and the cytochrome c oxidase. Since heme is an essential prosthetic group of many proteins engaged in the electron transfer, it is possible, that heme bound to the mitoBKCa channel can be involved in this interaction. However, the molecular basis of this putative phenomenon remains to be elucidated. The inhibition of mitoBKCa upon hemin binding can be a potential novel mechanism of neurotoxicity accompanying intracerebral hemorrhage. It is noteworthy, that hemin toxicity appears after quite a long time from the onset of the first clinical symptoms of ICH. Therefore, it would be theoretically feasible to implement an anti-hemin therapy early enough to prevent, or at least diminish its cytotoxicity. This could be accomplished either by the blockage of the hemin uptake by cells, or by the increase in the heme degradation rate. However, this issue still remains to be elucidated. Although encouraging, presented results shall be further verified in an experimental model of ICH. Acknowledgments This study was supported by the International PhD Studies in Neurobiology Program (MPD/2009/4) financed by the Foundation for Polish Science with the participation of the European Regional Development Funds. The support of Stiftung für medizinische Wissenschaft (Q-025.0205), Frankfurt am Main to W.S.K. is gratefully acknowledged. References Bednarczyk, P., Kowalczyk, J.E., Beresewicz, M., Dołowy, K., Szewczyk, A., Zabłocka, B., 2010. Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria. Biochem. Biophys. Res. Commun. 397, 614–620. http://dx.doi.org/10. 1016/j.bbrc.2010.06.011. Bednarczyk, P., Kozieł, A., Jarmuszkiewicz, W., Szewczyk, A., 2013a. Large conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells. Am. J. Physiol. Heart Circ. Physiol 304, 1415–1427. http://dx.doi.org/10.1152/ ajpheart.00976.2012. Bednarczyk, P., Wieckowski, M.R., Broszkiewicz, M., Skowronek, K., Siemen, D., Szewczyk, A., 2013b. Putative structural and functional coupling of the mitochondrial BKCa channel to the respiratory chain. PLoS One 8, e68125. http://dx.doi.org/10.1371/journal. pone.0068125. Cheng, Y., Gu, X.Q., Bednarczyk, P., Wiedemann, F.R., Haddad, G.G., Siemen, D., 2008. Hypoxia increases activity of the BK-channel in the inner mitochondrial membrane and reduces activity of the permeability transition pore. Cell. Physiol. Biochem. 127–136. Chen-Roetling, J., Regan, R.F., 2006. Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin. Biochem. Biophys. Res. Commun. 350, 233–237. http://dx.doi.org/10.1016/j.bbrc.2006.09.036. Chen-Roetling, J., Cai, Y., Lu, X., Regan, R.F., 2014. Hemin uptake and release by neurons and glia. Free Radic. Res. 48, 200–205. http://dx.doi.org/10.3109/10715762.2013. 859386. Dang, T.N., Bishop, G.M., Dringen, R., Robinson, S.R., 2010. The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin. Glia 58, 55–65. http://dx.doi.org/10.1002/glia.20901. Dang, T.N., Bishop, G.M., Dringen, R., Robinson, S.R., 2011a. The metabolism and toxicity of hemin in astrocytes. Glia 59, 1540–1550. http://dx.doi.org/10.1002/glia.21198. Dang, T.N., Robinson, S.R., Dringen, R., Bishop, G.M., 2011b. Uptake, metabolism and toxicity of hemin in cultured neurons. Neurochem. Int. 58, 804–811. http://dx.doi.org/10. 1016/j.neuint.2011.03.006.
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Regular Article
Hemin inhibits the large conductance potassium channel in brain mitochondria: A putative novel mechanism of neurodegeneration Bartłomiej Augustynek a,b, Alexei P. Kudin b, Piotr Bednarczyk a,c, Adam Szewczyk a,⁎, Wolfram S. Kunz b a b c
Laboratory of Intracellular Ion Channels, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland Department of Epileptology, University of Bonn, 25 Sigmund-Freud Street, D-53105 Bonn, Germany Department of Biophysics, Warsaw University of Life Sciences, 159 Nowoursynowska Street, 02-776 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 10 December 2013 Revised 14 April 2014 Accepted 23 April 2014 Available online 30 April 2014 Keywords: Mitochondria Hemin Intracerebral hemorrhage Mitochondrial BKCa channel Neurodegeneration ROS synthesis
a b s t r a c t Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca2+-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca2+-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca2+-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH. © 2014 Elsevier Inc. All rights reserved.
Introduction Intracerebral hemorrhage is a type of intracranial bleeding that occurs within the brain tissue. It can either be caused by brain trauma or occur in hemorrhagic stroke. Stroke is the second most common cause of death worldwide (Donnan et al., 2008), and hemorrhagic strokes account for 10–15% of all stroke cases (Sudlow and Warlow, 1997). Mortality of hemorrhagic stroke is high, and has not fallen within the last years. According to different studies it varies from 31% at 7 days to more than 90% at 10 years (Flaherty et al., 2006; Fogelholm et al., 2005). In hemorrhagic stroke the rapture or leak of a blood vessel leads to the formation of hematoma which oppresses the surrounding tissue and hampers their supply in oxygen and nutrients. Cells within and adjacent to the hematoma die quickly and there is not much that can be done today to prevent it. However, there is a pool of cells that experience negative effects of hemorrhage, although do not die immediately. Such cells would be a suitable target for potential therapies. It
⁎ Corresponding author. E-mail address: [email protected] (A. Szewczyk).
http://dx.doi.org/10.1016/j.expneurol.2014.04.022 0014-4886/© 2014 Elsevier Inc. All rights reserved.
is widely believed that one of the causes of cell loss in hemorrhagic stroke is the erythrocyte lysis that occurs in hematoma. Neurotoxic agents like hemoglobin, and its breakdown product hemin migrate from hematoma into surrounding tissue impairing the proper function of nervous cells (Chen-Roetling and Regan, 2006; Dang et al., 2011b). Hemin liberated from the hematoma can reach concentrations of as high as 10 mM (Robinson et al., 2009). Both astrocytes and neurons express heme carrier protein 1 (HCP1) in the plasma membrane, and hence can accumulate hemin upon its release from disintegrating erythrocytes (Dang et al., 2010, 2011b). Recent experimental data show rapid uptake of hemin by all brain resident cells, especially by microglia (Chen-Roetling et al., 2014). Additionally, hemin is a lipophilic compound that can intercalate into lipid bilayers, and cross them. However, one should expect that this phenomenon is rather of minor importance for hemin accumulation by cells. Still, it may play a role in hemin trafficking within the cell. Although neurotoxicity of hemin has been well documented, molecular basis of this phenomenon remains unclear. There are many processes and proteins which function can be impaired by this agent. One of them, the large-conductance Ca2 +-regulated potassium channel (BKCa channel) in the plasma membrane is inhibited by hemin (Tang et al., 2003).
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The BKCa channel is ubiquitously expressed in the plasma membrane of both excitable and non-excitable cells. It is activated by the changes in membrane potential and/or the calcium ions. Although it was extensively studied mainly in the plasma membrane, its counterparts exist within several intracellular structures like mitochondria (Siemen et al., 1999; Szabò et al., 2012). MitoBKCa is one of the best described channels present in the inner mitochondrial membrane. It can be blocked by charybdotoxin (ChTx), iberiotoxin (IbTx) or paxilline (Pax) and stimulated by calcium ions, changes in membrane potential, or by certain synthetic agents like NS1619. The large conductance calcium regulated potassium channel was identified in mitochondria of several cell types, namely: human glioma cell line LN229 (Siemen et al., 1999), guinea pig ventricular cells (Xu et al., 2002), skeletal muscle (Skalska et al., 2008), endothelial cells (Bednarczyk et al., 2013a), astrocytes (Thiede et al., 2012), astrocytoma (Bednarczyk et al., 2013b) and neurons (Fahanik-Babaei et al., 2011; Piwonska et al., 2008; Skalska et al., 2009). Although BKCa channels exist in many different locations throughout the cell, all of their forms share functional characteristics with their plasma membrane counterpart and all are encoded by a single gene: Kcnma1. Hence, it is widely believed that different types of BKCa channels are the results of alternative splicing of this gene. Although, the molecular identity of the mitoBKCa channel still remains to be elucidated, in 2013 Singh and co-workers have shown that a 50-aa C-terminal splice insert determines the mitochondrial location of mitoBKCa (Singh et al., 2013). Potassium channels are notorious for their role in cytoprotection (Escande and Cavero, 1992; Garlid, 2000; Malinska et al., 2010). Such cytoprotection could be especially important for tissues with a poor endogenous antioxidant defense such as the cardiac muscle or the nervous system where undisturbed and continuous blood flow and oxygen supply are extremely important (Doré, 2002). It is broadly accepted that preconditioning with the potassium channel openers (KCOs) results in cytoprotection throughout activation of mitochondrial channels (Facundo et al., 2006). The basis of KCO cytoprotective properties still remains to be elucidated, although it is suspected that attenuation of ROS synthesis in mitochondria that is observed after KCO administration may play some role in this phenomenon. Under substrate conditions that allow reverse electron flow, matrix K+ influx through the open mitoBKCa channel is believed to accelerate forward electron transfer and inhibit ROS production (Heinen et al., 2007a). Acceleration of ROS synthesis rate in turn is linked with several neurodegenerative diseases (Popa-Wagner et al., 2013). Another possible explanation of the cytoprotective properties of KCOs is mild uncoupling. The opening of mitochondrial potassium channels results in a modest dissipation of proton motive force by contributing inward current and following utilization of some of the proton gradient for K+ ejection from the matrix through the K+/H+ exchanger (O'Rourke, 2004) leading to decreased ROS synthesis (Kulawiak et al., 2008). The goal of this study was to elucidate whether hemin can inhibit the large conductance calcium regulated potassium channel in the mitochondrial inner membrane and if so, how does it alter some of the known beneficial effects of potassium channel openers on cell function.
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Cell culture and preparation of astrocytoma mitochondria Mitoplasts for patch-clamp experiments were prepared from the human astrocytoma U-87 MG cell line. The cell line identity was confirmed by the short tandem repeat (STR) profiling technique. This assay was performed according to the guidelines published by Masters et al. (2001). More details are in Bednarczyk et al. (2013b). For the experiments, cells were cultured in DMEM medium (10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin) at 37 °C in a humidified atmosphere with 5% CO2. The cells were fed and reseeded every third day. Once reached a confluence of about 70%, cells from five 75 cm2 flasks were scraped and centrifuged at 800 ×g for 10 min, resuspended in the preparation solution for the mitochondrial isolation (250 mM sucrose, 5 mM HEPES, pH = 7.2) and homogenized. First (9200 ×g, 10 min) and second (770 ×g, 10 min) centrifugation steps were performed to separate the fraction enriched in mitochondria. Sucrose was removed by two further fast centrifugation steps (9200 ×g, 10 min) in the storage solution (150 mM KCl, 10 mM HEPES, pH = 7.2). All procedures were performed at 4 °C. Patch clamp experiments Patch-clamp experiments on mitoplasts (spherical vesicles surrounded by the mitochondrial inner membrane) were performed as described previously (Bednarczyk et al., 2010, 2013b; Cheng et al., 2008; Toczyłowska-Mamińska et al., 2013). Briefly, a sample of purified human astrocytoma mitochondria was put into a hypotonic solution (5 mM HEPES, 200 μM CaCl2, pH = 7.2) for about 1 min in order to induce swelling and disruption of the mitochondrial outer membrane. Then, the addition of a hypertonic solution (750 mM KCl, 30 mM HEPES, 200 μM CaCl2, pH = 7.2) restored the isotonicity of the medium, and stopped further swelling. Mitoplasts were distinguished from the cellular debris present in preparation by their transparency, spherical shape, and characteristic ‘cap’ — remain of the outer membrane. In each experiment, one free-floating mitoplast was attached to the patch-clamp pipette made of borosilicate glass filled with an isotonic solution (150 mM KCl, 10 mM HEPES, 200 μM CaCl2, pH = 7.2). The isotonic solution was also used in control recordings for all presented data. Hemin and iberiotoxin (300 nM hemin and 2 nM IbTx in isotonic solution) were added from the back of the patch-clamp pipette through the peristaltic pump-driven capillary-pipe system. The experiments were carried out in patch-clamp inside-out mode. Reported voltages are those applied to the patch clamp pipette interior. The current was recorded by the patch-clamp amplifier Axopatch 200B (Molecular Devices Corporation). The pipettes had a resistance of 10–20 MΩ and were pulled by a Flaming/Brown type P-100 puller (Sutter Instrument). The measured ion currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. All experimental traces were recorded at the single-channel mode. The conductance was calculated from the current–voltage characteristics (data not shown). The probability of channel opening was determined using the singlechannel search mode of the Axon™ pCLAMP® 10 Electrophysiology Data Acquisition & Analysis Software (Molecular Devices Corporation).
Materials and methods
Isolation of rat brain mitochondria
All standard chemicals along with 1,3-dihydro-1-[2-hydroxy5(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), iberiotoxin (IbTx), charybdotoxin (ChTx), horseradish peroxidase (HRP) and hemin were purchased from Sigma-Aldrich. The uncoupler TTFB (4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole) was a kind gift from Prof. B. Beechey (Aberystwyth, UK). Catalase, digitonin and superoxide dismutase (SOD) were purchased from Serva, and bacterial proteinase from Fluka. All cell culture materials were purchased from Gibco.
Solutions used for: mitochondrial isolation: MSE solution (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES, 1 mg/ml essential fatty acid free BSA, pH = 7.4); measurements: K+ containing MTP medium (10 mM KH2PO4, 60 mM KCl, 60 mM Tris–HCl, 110 mM mannitol, 5 mM MgCl2, 0.5 mM EDTA, pH = 7.4) and K+ free MTP medium (10 mM NaH2PO4, 60 mM NaCl, 60 mM Tris–HCl, 110 mM mannitol, 5 mM MgCl2, 0.5 mM EDTA, pH = 7.4). Rat brain mitochondria were isolated according to the standard protocol described by Rosenthal et al. (1987), with a small modification
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that allowed obtaining mitochondria with much better functional characteristics (Kudin et al., 2004).
significance of the obtained results. P b 0.05 was considered statistically significant. Significance levels are: 0.05–0.01 (*), 0.01–0.001 (**), and b0.001 (***).
Measurements of H2O2 generation by rat brain mitochondria Results In the presence of HRP H2O2 oxidizes colorless p-hydroxyphenylacetic acid (pHPPA, 200 μM). This reaction results in the formation of its fluorescent dimer (pHPPA)2 (λex = 317 nm, λem = 414 nm). All measurements were performed with a RF-5001 spectrofluorophotometer (Shimadzu). Shortly, mitochondrial H2O2 generation was fueled by succinate (10 mM) and measured at 30 °C in an oxygen-saturated MTP medium. The reactions were catalyzed by HRP (20 U/ml) in the presence of mitochondria (0.2 mg of protein/ml). An excess of SOD (176 U/ml) guaranteed the complete conversion of all produced superoxide into hydrogen peroxide (Kudin et al., 2004; Malinska et al., 2009). The fluorescence signal was calibrated by a series of hydrogen peroxide additions (normally 6 additions of 140 pmol H2O2) in the presence of pHPPA and isolated mitochondria. Measurements of rat brain mitochondrial membrane potential Changes in the potential of the mitochondrial inner membrane (ΔΨm) were evaluated by the rhodamine 123 (2.6 μM) fluorescence quenching (λex = 450 nm, λem = 550 nm), measured with a RF-5001 spectrofluorophotometer (Shimadzu). The measurements were performed in MTP media at 30 °C. 10 mM glutamate and 5 mM malate were used to energize mitochondria. The concentration of rat brain mitochondrial protein was about 0.2 mg/ml. Addition of mitochondria causes a sharp drop in the rhodamine fluorescence. Minimal fluorescence corresponds to the maximal membrane potential. Any increase of the rhodamine fluorescence means dissipation of the ΔΨm. To calculate the mitochondrial membrane potential, the fluorescence changes were calibrated with corresponding potassium diffusion potentials. Briefly, 100 μM valinomycin was added to the rotenone de-energized mitochondrial preparation and incubated in a medium in which K+ ions were replaced by Na+. Next, the preparation was titrated with precisely defined additions of KCl until no further changes of rhodamine 123 fluorescence were recorded (equilibrium point). The corresponding potentials were calculated using the Nernst equation. Data analysis All experimental values are reported as mean ± SD (standard deviation). The Student's t test was used to determine the statistical
Patch-clamp Patch-clamp experiments were performed on mitoplasts obtained from the human astrocytoma U-87 MG cell line. All currents were measured in symmetric 150/150 mM KCl isotonic solution with 200 μM CaCl2. To determine whether the observed channel activity was associated with the BKCa we reduced the Ca2 + concentration from high (200 μM) to low (1 μM) and tested its sensitivity to the BKCa inhibitors. All low calcium conditions, iberiotoxin and paxilline inhibited channel activity (Bednarczyk et al., 2013b). Furthermore, at the end of each experiment, an addition of iberiotoxin (100 nM) was used to confirm the identity of the channel (immediate channel inhibition). Fig. 1A shows the single-channel recordings of mitoBKCa activity at different voltages in control conditions and after addition of 300 nM hemin. In Fig. 1B we present results from 9 experiments of such type in the context of channel activity, each comprising 1 min of recordings. This analysis revealed that hemin inhibits the activity of the channel by about 40% at +40 mV, and this inhibition is statistically significant. H2O2 synthesis in rat brain mitochondria In the current study we have measured succinate fueled H2O2 generation rate in the conditions that allow reverse electron flow. As it was previously shown, in these conditions addition of BKCa channel openers, such as NS1619, causes substantial attenuation of H2O2 generation rate (Kulawiak et al., 2008). According to our measurements, NS1619 (2.5 μM) attenuates brain mitochondrial ROS generation rate by reverse and forward electron flow by about 45%. As visible in Fig. 2A the addition of hemin (2.5 μM) inhibits this effect by about 30%. It is noteworthy, that the strength of this inhibition is comparable with that caused by iberiotoxin (5 μM) (Fig. 2B). Each experiment was finalized by an addition of catalase to inhibit further H2O2 generation. As one can see, the effectiveness of hemin/iberiotoxin attenuation of NS1619 effect was limited. It can be explained by the fact that a decrease in H2O2 synthesis rate caused by NS1619 comes from a combination of its specific properties towards the mitoBKCa channel and some unspecific actions. Therefore, hemin along with iberiotoxin abolishes probably only a part of the observed effect that results from inhibition of the mitoBKCa channel.
Fig. 1. Single channel recordings of the mitoBKCa channel in the presence of 300 nM hemin. A. Raw experimental traces (symmetric 150/150 mM KCl isotonic solution with 200 μM Ca2+) at different voltages, ‘–’ indicates closed state. B. Average probability of opening of the mitoBKCa channel upon addition of 300 nM hemin, n = 9.
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Fig. 2. ROS synthesis rate. A. Representative experimental traces showing the effect of: 2.5 μM NS1619, 2.5 μM NS1619 + 2.5 μM hemin and 2.5 μM NS1619 + 5 μM iberiotoxin on the H2O2 synthesis rate by isolated rat brain mitochondria (10 mM succinate, 200 μM pHPPA, 20 U/ml HRP, 176 U/ml SOD). AF — arbitrary fluorescence units. B. Average H2O2 synthesis rate after administration of 2.5 μM NS1619, 2.5 μM NS1619 + 2.5 μM hemin and 2.5 μM NS1619 + 5 μM iberiotoxin, n = 7.
The remaining (hemin/iberiotoxin-insensitive) NS1619 effect is likely caused by the unspecific properties of the opener (Debska et al., 2003; Heinen et al., 2007b; Kicinska and Szewczyk, 2004). Dissipation of mitochondrial inner membrane potential To investigate the possible impact of hemin on mitochondrial membrane potential throughout activation of the mitoBKCa channel we have performed a set of experiments employing fluorescent properties of rhodamine 123. The samples of rat brain mitochondria oxidizing glutamate and malate (10 mM, 5 mM respectively) were treated with NS1619 (2.5 μM). This resulted in a mild membrane potential dissipation (about 12 mV) (Fig. 3A). In the presence of 2.5 μM hemin (Fig. 3B), and 2.5 μM charybdotoxin (Fig. 3C), the effect of NS1619 on
membrane potential was substantially attenuated. Each experiment was completed by an addition of the uncoupler (TTFB, 1 μM) to deenergize the mitochondria. To prove that the observed effect is specific to potassium ion permeability, we have performed control experiments in a potassium free medium. In the absence of potassium ions, the reported influence of NS1619 on the membrane potential was comparable with the one observed after ChTx addition (Fig. 3D). We have also verified the relationship between concentration of hemin and its inhibitory effect on the opening of the mitoBKCa channel (Fig. 4). Hemin clearly acts in a dose-dependant manner. However, in these conditions the increase of dose above 3 μM does not significantly alter membrane potential any longer. Taken together, hemin can abolish about 50% of the NS1619 potential-dissipating properties.
Fig. 3. Changes in the membrane potential. A, B, C, D — Representative experimental traces for isolated brain mitochondria upon addition of 2.5 μM NS1619. The fluorescence of rhodamine 123 is inversely proportional to the membrane potential. mito — addition of isolated brain mitochondria; TTFB — uncoupler (1 μM), and AF — arbitrary fluorescence units.
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Fig. 4. Hemin dose response. Dissipation of the mitochondrial inner membrane potential upon addition of 2.5 μM NS1619 in the presence of increasing concentrations of hemin (1–4 μM), n = 12.
Discussion In 2003, Tang and co-workers have shown that heme and the product of its oxidation hemin can bind to, and inhibit the BKCa channel in the plasma membrane (Tang et al., 2003). Since an increase in the potassium ion permeability of the mitochondrial inner membrane contributes to cytoprotection (O'Rourke, 2004; Xu et al., 2002), it is possible that the phenomenon of mitoBKCa channel inhibition by hemin could exert some negative impact on cells. This effect would be particularly pronounced in the case of massive hemin influx which occurs in neurological conditions like intracerebral and subarachnoid hemolysis, hemorrhagic stroke or brain trauma. As it was previously mentioned, brain hemorrhages are potent sources of hemin that infiltrates to the interstitial space within brain tissue, and literally floods neurons, astrocytes and other brain cells. The issue of hemin toxicity in hemorrhagic stroke was comprehensively reviewed by Robinson et al. (2009). According to this group, neurons accumulate hemin 50% faster than astrocytes, and therefore are more susceptible to its cytotoxic properties. Moreover, they suggested that the extent of hemin toxicity cannot be simply explained by the mechanisms described so far (Dang et al., 2011a,b) and concluded that the molecular basis of hemin neurotoxicity appears to be multilevel, thus requires more attention. It has been previously proposed that the BKCa channel posses a highly conserved heme binding motive, the same as described in cytochrome c (Jaggar et al., 2005; Tang et al., 2003). According to the mechanism postulated by Jaggar and co-workers reduced heme is a physiological inhibitor of BKCa channels. It has the ability to bind carbon monoxide (CO) a molecule that is constantly produced in very small quantities by heme oxygenases. According to this model, heme can be considered as a cellular CO sensor. Once CO binds to the heme that is attached to a BKCa channel molecule it alters heme–channel interaction that results in its activation. In light of these reports inhibitory effect of hemin can rely on the replacing of the reduced heme molecules by oxidized hemin. Hemin cannot bind CO; hence it maintains the channel in the closed state. This model seems to be even more valid when we consider data published by Yi and co-workers in 2010. According to them, the BKCa channel can directly interact with heme oxygenase-2 (a CO producer). They have described a sophisticated thiol/disulfide switch in the heme binding domain of the channel, claiming that this is the mechanism by which the activity of the BKCa channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions (Yi et al., 2010). Summarizing, the strong evidences prove that the BKCa channel along with heme oxygenase, heme and CO can take part in a sophisticated mechanism that allow the cell to detect changes in its internal redox state. It is possible that similar mechanisms regulate the mitoBKCa channel. Mitochondria are the cellular power plants relying on continuous redox reactions that drive the ATP synthesis. Therefore, it seems
likely that a redox state-sensitive ion channel (e.g. mitoBKCa) could play an important role in the proper function of mitochondria. The results presented in the current work provided to our knowledge the first proof that hemin can inhibit the mitoBKCa channel. This in turn diminishes the beneficial effects of mitoBKCa channel opening such as dissipation of the membrane potential and the concomitant decrease in ROS synthesis rate. According to recently published work (Bednarczyk et al., 2013b), there is a putative structural and functional coupling of the mitoBKCa channel with the elements of the mitochondrial respiratory chain in the human astrocytoma U-87 MG cell line. It appeared that acceleration of the electron transfer by addition of its substrates inhibits activity of the mitoBKCa channel and this effect can be abolished by the inhibitors of the respiratory chain. It was showed that there is a putative interaction between the regulatory subunit of the mitoBKCa channel and the cytochrome c oxidase. Since heme is an essential prosthetic group of many proteins engaged in the electron transfer, it is possible, that heme bound to the mitoBKCa channel can be involved in this interaction. However, the molecular basis of this putative phenomenon remains to be elucidated. The inhibition of mitoBKCa upon hemin binding can be a potential novel mechanism of neurotoxicity accompanying intracerebral hemorrhage. It is noteworthy, that hemin toxicity appears after quite a long time from the onset of the first clinical symptoms of ICH. Therefore, it would be theoretically feasible to implement an anti-hemin therapy early enough to prevent, or at least diminish its cytotoxicity. This could be accomplished either by the blockage of the hemin uptake by cells, or by the increase in the heme degradation rate. However, this issue still remains to be elucidated. Although encouraging, presented results shall be further verified in an experimental model of ICH. Acknowledgments This study was supported by the International PhD Studies in Neurobiology Program (MPD/2009/4) financed by the Foundation for Polish Science with the participation of the European Regional Development Funds. The support of Stiftung für medizinische Wissenschaft (Q-025.0205), Frankfurt am Main to W.S.K. is gratefully acknowledged. References Bednarczyk, P., Kowalczyk, J.E., Beresewicz, M., Dołowy, K., Szewczyk, A., Zabłocka, B., 2010. Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria. Biochem. Biophys. Res. Commun. 397, 614–620. http://dx.doi.org/10. 1016/j.bbrc.2010.06.011. Bednarczyk, P., Kozieł, A., Jarmuszkiewicz, W., Szewczyk, A., 2013a. Large conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells. Am. J. Physiol. Heart Circ. Physiol 304, 1415–1427. http://dx.doi.org/10.1152/ ajpheart.00976.2012. Bednarczyk, P., Wieckowski, M.R., Broszkiewicz, M., Skowronek, K., Siemen, D., Szewczyk, A., 2013b. Putative structural and functional coupling of the mitochondrial BKCa channel to the respiratory chain. PLoS One 8, e68125. http://dx.doi.org/10.1371/journal. pone.0068125. Cheng, Y., Gu, X.Q., Bednarczyk, P., Wiedemann, F.R., Haddad, G.G., Siemen, D., 2008. Hypoxia increases activity of the BK-channel in the inner mitochondrial membrane and reduces activity of the permeability transition pore. Cell. Physiol. Biochem. 127–136. Chen-Roetling, J., Regan, R.F., 2006. Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin. Biochem. Biophys. Res. Commun. 350, 233–237. http://dx.doi.org/10.1016/j.bbrc.2006.09.036. Chen-Roetling, J., Cai, Y., Lu, X., Regan, R.F., 2014. Hemin uptake and release by neurons and glia. Free Radic. Res. 48, 200–205. http://dx.doi.org/10.3109/10715762.2013. 859386. Dang, T.N., Bishop, G.M., Dringen, R., Robinson, S.R., 2010. The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin. Glia 58, 55–65. http://dx.doi.org/10.1002/glia.20901. Dang, T.N., Bishop, G.M., Dringen, R., Robinson, S.R., 2011a. The metabolism and toxicity of hemin in astrocytes. Glia 59, 1540–1550. http://dx.doi.org/10.1002/glia.21198. Dang, T.N., Robinson, S.R., Dringen, R., Bishop, G.M., 2011b. Uptake, metabolism and toxicity of hemin in cultured neurons. Neurochem. Int. 58, 804–811. http://dx.doi.org/10. 1016/j.neuint.2011.03.006.
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