In the present study, we describe the existence of a novel potassium channel in the plant [potato (Solanum tuberosum) tuber] mitochondrial inner membrane. We found that substances known to modulate large-conductance calcium-activated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca2+ and NS1619 {1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-ben-zimidazole-2-one; a potassium channel opener} were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilayers. After incorporation in 50/450 mM KCl gradient solutions, we recorded large-conductance potassium channel activity with conductance from 502±15 to 615±12 pS. The probability of channel opening was increased by Ca2+ and reduced by iberiotoxin. Immunological analysis with antibodies raised against the mammalian plasma-membrane large-conductance Ca2+-dependent K+ channel identified a pore-forming α subunit and an auxiliary β2 subunit of the channel in potato tuber mitochondrial inner membrane. These results suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

INTRODUCTION

Mitochondria play an important role in energy metabolism within the cell. In addition to this canonical function, mitochondria are involved in intracellular signalling. Recently, potassium transport through the mitochondrial inner membrane was found to play a central role in the cytoprotection of various mammalian cell types [1,2]. This transport is strictly ion-channel-dependent. Similarly to the plasma membrane, potassium-selective ion channels are found in the mitochondrial inner membrane [35]. Potassium ions control mitochondrial metabolism, primarily via a regulation of matrix volume [6]. The biophysical and pharmacological properties of mitochondrial potassium channels are similar to some of the potassium channels present in the plasma membrane of various mammalian cell types, for example mitoKATP channels (mitochondrial ATP-sensitive potassium channels) [7], mitoBKCa channels (mitochondrial large-conductance Ca2+-activated potassium channels) [8] and voltage-dependent potassium Kv1.3 channels [9].

In plant mitochondria, the existence of different energy-dissipating systems is necessary for metabolic regulation [10]. Plant mitochondria (but not animal mitochondria) contain AOX, an alternative oxidase that catalyses ubiquinol:oxygen oxidoreduction without H+ release into the cytosol, thus dissipating the redox potential energy [11,12]. Similarly to animal mitochondria, plant mitochondria contain PUMP (plant uncoupling mitochondrial protein), which dissipates a proton electrochemical gradient by mediating re-entry of H+ into the mitochondrial matrix in the presence of unbound fatty acids [13]. Another energy-dissipating system present in plant mitochondria (as in animal mitochondria) is the ATP-sensitive potassium channel mitoKATP, which mediates an electrophoretic K+ uniport, probably working together with an electroneutral K+/H+ exchanger [1417]. Descriptions of the plant mitoKATP channel have been based mainly on studies of isolated mitochondria in which the influence of K+ entry into the mitochondrial matrix on membrane potential (ΔΨ) dissipation and mitochondrial swelling were assessed. The plant mitoKATP channel seems to be sensitive to the same potassium channel modulators as the mammalian mitoKATP channel. In durum wheat (Triticum durum) and pea (Pisum sativum) mitochondria, the mitoKATP channel is activated by diazoxide and GTP, and its activity is inhibited by glyburide and 5-hydroxydecanoate (in pea mitochondria) [14,16,18]. Moreover, the plant mitoKATP appears to be stimulated by cyclosporin A and regulated by dithiol-disulfide interconversion, H2O2 and NO [15,16,19]. On the other hand, an ATP-insensitive, quinine-inhibited potassium import pathway has been described in potato (Solanum tuberosum), tomato (Solanum lycopersicon) and maize (Zea mays) mitochondria [20].

So far, there has been no information about other possible plant mitochondrial potassium channels, such as the mitoBKCa channel described in animal mitochondria. Therefore, the aim of the present study was to search for a mitoBKCa channel in plant mitochondria and to determine the bioenergetic consequences of its activation. For this purpose, we studied the effects of BKCa channel (plasma membrane large-conductance Ca2+-activated potassium channel) activators {Ca2+, NS1619 1,3-dihydro1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2Hbenzimidazole-2-one} and inhibitor [IbTx (iberiotoxin)] on ΔΨ and respiration in isolated potato tuber mitochondria. Moreover, measurements of the electrophysiological properties of a single mitoBKCa channel in a reconstituted system were carried out and immunological detection experiments were performed to confirm the discovery of a plant mitoBKCa channel.

EXPERIMENTAL

Chemicals

L-α-Phosphatidylcholine (as asolectin), NS1619 and n-decane were purchased from Sigma–Aldrich, and IbTx was from Bachem. All other chemicals were of the highest purity commercially available. NS1619 was dissolved in methanol, and IbTx in water.

Isolation of mitochondria

Potato tubers were purchased from the local supermarket. Peeled potato tubers (1 kg) were homogenized in a blender in 1 litre of ice-cold medium consisting of 0.35 M mannitol, 3 mM EDTA, 3 mM EGTA, 6 mM cysteine, 0.1% BSA and 25 mM NaH2PO4/Na2HPO4 buffer. The pH was adjusted to 8.0 with NaOH. The crude extract was quickly filtered through cheesecloth, and the filtrate was centrifuged at 1000 g for 10 min. The supernatant was further centrifuged at 11000 g for 20 min. The pellet of crude mitochondria was washed with medium consisting of 0.35 M mannitol, 1.5 mM EGTA, 0.1% BSA and 10 mM NaH2PO4/Na2HPO4 buffer, pH 6.8, and centrifuged again at 1000 g for 5 min and then at 11000 g for 20 min. Mitochondria were subsequently purified on a self-generating 22% Percoll gradient (at 40000 g for 30 min). Purified mitochondria were washed twice in the same medium without EGTA and BSA. The mitochondrial protein concentration was determined by the biuret method.

Preparation of membrane fractions

To obtain SMPs (submitochondrial particles), mitochondria were isolated as above, but with media containing potassium salts. Freshly prepared mitochondria were diluted to approx. 8 mg of protein/ml with 0.3 M sucrose and 10 mM Hepes/KOH, pH 7.2, and subsequently frozen at −20 °C. After thawing, the suspension was sonicated six times for 30 s using sonifier UD-11 (TechPan, Poland) at its maximal output. Then, the suspension was centrifuged at 12000 g for 15 min to pellet the unbroken mitochondria. The supernatant was centrifuged at 200000 g (2 h) to pellet SMPs. Fractions of SMPs, 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.

To obtain a plasma-membrane-enriched fraction, the supernatant from the first 11000 g centrifugation during mitochondria isolation was centrifuged at 100000 g for 1h. The pellet was resuspended in 0.3 M sucrose and 10 mM Hepes/KOH, pH 7.2.

PLM (planar lipid membrane) measurements

Experiments were performed with potato tuber SMPs, as described previously [2123]. In brief, PLMs were formed in a 250-μm-diameter hole drilled in a Delrin cup (Warner Instruments), which separates the two chambers (cis and trans, each with a 1 ml internal volume). The chambers contained 50/450 mM KCl (trans/cis) and 20 mM Tris/HCl solutions, 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. PLMs were painted using asolectin in n-decane at a final concentration of 25 mg of lipid/ml. SMPs (approx. 3 μg of protein/ml, 0.5–1.5 μl/reconstitution) were added to the trans compartment (see Figure 5A). All measurements were carried out at room temperature (~25 °C). Formation and thinning of the bilayer were monitored by capacitance measurements and optical observations. Final accepted capacitance values ranged from 120 to 230 pF. Electrical connections were created using 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. The current was measured using a bilayer membrane amplifier (BLM-120; BioLogic). Signals were low-pass-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 personal computer for off-line analysis by Chart version 5.5.5 (PowerLab ADInstruments) and pCLAMP10.2 (Axon Instruments). The pCLAMP10.2 software package was used for data processing. The channel recordings presented are representative of the most frequently observed conductance values under the given conditions. Ion conductance was calculated from the linear regression of the points in the current–voltage relationship using the GraphPad Prism 4 program.

Mitochondrial oxygen consumption

Oxygen uptake was measured polarographically with a Clark-type oxygen electrode (Rank Brothers) in 3 ml of standard incubation medium (25 °C) consisting of 0.35 M mannitol, 3 mM NaH2PO4, 1 mM MgCl2, 10 mM Tris/HCl, 10 mM Mes, pH 6.8, and 0.1% BSA. Changes in the composition of the incubation medium are described in the Figure legends. Measurements were performed with 0.8–1 mg of mitochondrial protein in the presence of 1.5 mM benzohydroxymate (an inhibitor of AOX), 0.14 mM ATP (to activate succinate dehydrogenase), 1.8 μM carboxyatractylozide (to exclude ATP/ADP antiporter activity) and 10 mM glibenclamide (to inhibit the mitoKATP channel). Succinate (5 mM) plus rotenone (2 μM) was used as a respiratory substrate. Respiratory rate measurements were performed in the absence of added ADP, i.e. in the resting state (State 4). State 3 (phosphorylating) respiration measurements were performed 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 2.5–3 (with succinate), were used in all experiments. O2 uptake values are presented in nmol of O·min−1·mg of protein−1.

Mitochondrial-membrane-potential measurements

The ΔΨ was measured simultaneously with oxygen uptake using a TPP+ (tetraphenylphosphonium)-specific electrode as described by Kamo et al. [24]. Measurements were performed in the presence of 6 μM TPP+. To calculate the ΔΨ value, the matrix volume of potato tuber mitochondria was assumed to be 2.0 μl·mg of protein−1. The calculation assumes that the TPP+ distribution between mitochondria and medium followed the Nernst equation. Corrections were made for TPP+ binding to mitochondrial membranes. Values of ΔΨ are presented in mV.

SDS/PAGE and immunoblotting

Samples of isolated mitochondrial proteins, SMPs or the plasma-membrane-enriched fraction were solubilized in sample buffer containing 2% (w/v) SDS, 50 mM Tris/HCl (pH.6.8), 10% (v/v) glycerol, 0.004% (w/v) Bromophenol Blue and 8% mercaptoethanol, and then boiled for 4 min. Proteins were separated in SDS/12.5% polyacrylamide gels, and then electrotransferred to a nitrocellulose membrane. Membranes were then hybridized with anti-KCa1.1 and anti-sloβ2 antibodies (APC-107 and APC-034; Alomone) at dilutions of 1:500 or 1:200 respectively, in the presence or absence of blocking peptide. Cross-reactivity was also checked with antibodies raised against a plant plasma-membrane H+-ATPase (at a dilution of 1:1000). Protein detection was achieved with secondary antibodies linked to horseradish peroxidase (at a dilution of 1:20000) and the ECL® (enhanced chemiluminescence) system. Protein content was determined by the Bradford method using Bio-Rad equipment.

RESULTS

Effect of potassium-channel modulators on respiratory rate and membrane potential in isolated potato tuber mitochondria

To investigate the possible effects of Ca2+ on the potassium permeability of isolated potato tuber mitochondria, we measured mitochondrial resting (non-phosphorylating) respiration and ΔΨ in potassium-containing medium in the presence of different Ca2+ concentrations, using succinate as an oxidizable substrate (Figure 1). An example of simultaneous measurements of the respiratory rate and ΔΨ is shown in Figure 1(A). Addition of Ca2+ up to a concentration of 1 mM caused an increased respiration rate of up to 20±2% in the absence of 1.5 μM IbTx, and up to 5±3% in its presence (n=4, mean±S.D.) (Figure 1C). At the same time, ΔΨ decreased after addition of Ca2+ by up to 6.8±0.5 mV and 0.7±0.4 mV in the absence and presence of IbTx respectively (n=4, mean±S.D.) (Figure 1D). Thus, the inhibitor previously described as specific for the mammalian large-conductance potassium channel [1,2] considerably blocks Ca2+-induced respiration and ΔΨ depolarization in isolated potato tuber mitochondria. These results suggest that Ca2+ stimulates IbTx-sensitive K+ flux into potato tuber mitochondria, decreasing ΔΨ and thus accelerating the mitochondrial respiration rate. This indicates that the mitoBKCa channel is present in potato tuber mitochondria. The Ca2+-induced IbTx-sensitive respiratory rate (i.e. the difference between the respiratory rate in the presence of Ca2+ and the rate in the presence of Ca2+ plus inhibitor) should represent mitoBKCa channel activity (i.e. the channel-mediated K+ flux). This activity, as measured using various Ca2+ concentrations (Figure 1B), revealed that 50% of the maximal stimulation (S0.5) by Ca2+ is reached at 0.45 mM, as calculated from the linear regression of a double-reciprocal plot (Figure 1B, inset). A similar value was attained when the Ca2+-induced IbTx-sensitive depolarization of ΔΨ compared with the Ca2+ concentration was analysed (results not shown). The partial insensitivity of Ca2+-induced respiration and ΔΨ depolarization to IbTx observed in potato tuber mitochondria is probably related to electrophoretic Ca2+ influx pathways present in some plant mitochondria [25] or permeability transition opening [26].

Iberiotoxin-sensitive influence of CaCl2 on resting respiratory rate and membrane potential

Figure 1
Iberiotoxin-sensitive influence of CaCl2 on resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. (A) Succinate (5 mM), 1.5 μM IbTx and successive doses of CaCl2 were added as indicated. The broken trace shows the measurement obtained in the absence of Ca2+. An example of three measurements (using mitochondria from three different preparations) is shown. Numbers on the traces refer to O2 consumption rates in nmol of O·min−1·mg of protein−1 or to ΔΨ values in mV. (B) Ca2+-induced IbTx-sensitive respiration against CaCl2 concentration. Inset: double-reciprocal plot. (C and D) Effect of CaCl2 concentration on O2 consumption (C) and ΔΨ (D) in the presence or absence of 1.5 μM IbTx. Measurements were performed as in (A), except that IbTx was added prior to addition of Ca2+ where indicated. The results are representative of three different mitochondrial preparations (mean±S.D.). (C) the percentage of control respiration in the absence of modulators (119±4 nmol of O·min−1·mg of protein−1). (D) the change in ΔΨ induced by a given concentration of CaCl2 relative to the initial state 4 ΔΨ (216±1 mV, mean±S.D.) in the absence of modulators.

Figure 1
Iberiotoxin-sensitive influence of CaCl2 on resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. (A) Succinate (5 mM), 1.5 μM IbTx and successive doses of CaCl2 were added as indicated. The broken trace shows the measurement obtained in the absence of Ca2+. An example of three measurements (using mitochondria from three different preparations) is shown. Numbers on the traces refer to O2 consumption rates in nmol of O·min−1·mg of protein−1 or to ΔΨ values in mV. (B) Ca2+-induced IbTx-sensitive respiration against CaCl2 concentration. Inset: double-reciprocal plot. (C and D) Effect of CaCl2 concentration on O2 consumption (C) and ΔΨ (D) in the presence or absence of 1.5 μM IbTx. Measurements were performed as in (A), except that IbTx was added prior to addition of Ca2+ where indicated. The results are representative of three different mitochondrial preparations (mean±S.D.). (C) the percentage of control respiration in the absence of modulators (119±4 nmol of O·min−1·mg of protein−1). (D) the change in ΔΨ induced by a given concentration of CaCl2 relative to the initial state 4 ΔΨ (216±1 mV, mean±S.D.) in the absence of modulators.

The potassium channel opener NS1619, previously described to be specific for the mitoBKCa channel [1,2], was also used in our studies. Similarly to calcium ions, NS1619 stimulates mitochondrial resting oxygen uptake and decreases mitochondrial resting ΔΨ (Figure 2). An example of the effect of increasing concentrations of NS1619 on respiratory rate and ΔΨ is shown in Figure 2(A). Addition of NS1619 up to 40 μM resulted in an increase in the rate of respiration up to 100±6% and 53±7% (n=6, mean±S.D.) in the absence and presence of 1.5 μM IbTx respectively (Figure 2C). At the same time, ΔΨ decreased after addition of up to 40 μM NS1618 by up to 18.1±2 mV and 10.6±1.3 mV (n=6, mean±S.D.) in the absence and presence of IbTx respectively (Figure 2D). Thus, IbTx partially blocks (by approx. 50%) NS1619-induced respiration and ΔΨ depolarization in isolated potato tuber mitochondria. When compared with the effect of IbTx on Ca2+-induced respiration and ΔΨ depolarization (Figure 3), this could indicate a non-specific uncoupling effect of NS1619 on isolated potato tuber mitochondria rather than low sensitivity of the channel to IbTx. Similarly, the sensitivity of NS1619-induced respiration and ΔΨ depolarization to the mitoBKCa channel blockers (IbTx, charybdotoxin and paxilline) in some mammalian isolated mitochondria is not complete [2730]. Nevertheless, we can conclude that, in potato tuber mitochondria, NS1619 stimulates IbTx-sensitive K+ flux, decreases ΔΨ, and thus accelerates the mitochondrial respiration rate, further indicating the presence of the mitoBKCa channel activity. The NS1619-induced IbTx-sensitive respiratory rate (i.e. the difference between the respiratory rate in the presence of the modulator and the rate after addition of the inhibitor) measured at various NS1619 concentrations revealed that 50% of maximal stimulation (S0.5) is reached at 25 μM NS1619 (Figure 2B, inset). When NS1619-induced IbTx-sensitive depolarization of ΔΨ at various NS1619 concentrations was plotted, a similar value of S0.5 for NS1619 was obtained (results not shown).

Influence of NS1619, a mitoBKCa channel activator, on resting respiratory rate and membrane potential

Figure 2
Influence of NS1619, a mitoBKCa channel activator, on resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. (A) Succinate (5 mM), 1.5 μM IbTx, and successive doses of NS1619 were added as indicated. The broken trace shows the measurement obtained in the absence of activator. Numbers on the traces refer to O2 consumption rates in nmol of O·min−1·mg of protein−1 or to ΔΨ values in mV. (B) NS1619-induced IbTx-sensitive respiration against NS1619 concentration. Inset: double-reciprocal plot. (C and D) Effect of NS1619 concentration on O2 consumption (C) and ΔΨ (D) in the presence or absence of 1.5 μM IbTx. Measurements were performed as in (A), except that IbTx was added prior to activator addition where indicated. The data are representative of three different mitochondrial respirations (±S.D). (C) Percentage of control respiration in the absence of modulators (122±3 nmol of O·min−1·mg of protein−1). (D) Change in ΔΨ induced by a given concentration of CaCl2 relative to the initial state 4 ΔΨ (217±1 mV, ±S.D.) in the absence of modulators.

Figure 2
Influence of NS1619, a mitoBKCa channel activator, on resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. (A) Succinate (5 mM), 1.5 μM IbTx, and successive doses of NS1619 were added as indicated. The broken trace shows the measurement obtained in the absence of activator. Numbers on the traces refer to O2 consumption rates in nmol of O·min−1·mg of protein−1 or to ΔΨ values in mV. (B) NS1619-induced IbTx-sensitive respiration against NS1619 concentration. Inset: double-reciprocal plot. (C and D) Effect of NS1619 concentration on O2 consumption (C) and ΔΨ (D) in the presence or absence of 1.5 μM IbTx. Measurements were performed as in (A), except that IbTx was added prior to activator addition where indicated. The data are representative of three different mitochondrial respirations (±S.D). (C) Percentage of control respiration in the absence of modulators (122±3 nmol of O·min−1·mg of protein−1). (D) Change in ΔΨ induced by a given concentration of CaCl2 relative to the initial state 4 ΔΨ (217±1 mV, ±S.D.) in the absence of modulators.

Influence of iberiotoxin, a mitoBKCa channel inhibitor, on resting respiratory rate and membrane potential

Figure 3
Influence of iberiotoxin, a mitoBKCa channel inhibitor, on resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. (A) Succinate (5 mM), 40 μM NS1619, and successive doses of 0.5 μM IbTx, were added as indicated. The broken trace shows the measurement in the absence of IbTx addition. Numbers on the traces refer to O2 consumption rates in nmol of O·min−1·mg of protein−1 or to ΔΨ values in mV. (B) IbTx-sensitive NS1619-induced respiration versus the inhibitor concentration. Inset, double reciprocal plot. (C and D) Effect of IbTx concentration on O2 consumption (C) and ΔΨ (D) in the presence or absence of 0.8 mM CaCl2 or 40 μM NS1619. Measurements were performed as in (A). The data deal with three different mitochondrial respirations (mean±S.D.). (C) Percentage of control respiration in the absence of activators and IbTx (120±4 nmol of O·min−1·mg of protein−1). (D) Change in ΔΨ induced by a given modulator relative to the initial State 4 ΔΨ (216±1 mV, mean±S.D.).

Figure 3
Influence of iberiotoxin, a mitoBKCa channel inhibitor, on resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium additionally containing 50 mM KCl. (A) Succinate (5 mM), 40 μM NS1619, and successive doses of 0.5 μM IbTx, were added as indicated. The broken trace shows the measurement in the absence of IbTx addition. Numbers on the traces refer to O2 consumption rates in nmol of O·min−1·mg of protein−1 or to ΔΨ values in mV. (B) IbTx-sensitive NS1619-induced respiration versus the inhibitor concentration. Inset, double reciprocal plot. (C and D) Effect of IbTx concentration on O2 consumption (C) and ΔΨ (D) in the presence or absence of 0.8 mM CaCl2 or 40 μM NS1619. Measurements were performed as in (A). The data deal with three different mitochondrial respirations (mean±S.D.). (C) Percentage of control respiration in the absence of activators and IbTx (120±4 nmol of O·min−1·mg of protein−1). (D) Change in ΔΨ induced by a given modulator relative to the initial State 4 ΔΨ (216±1 mV, mean±S.D.).

Figure 3 illustrates the efficiency of IbTx in the inhibition of mitoBKCa channel activity under unstimulated (no activators) or stimulated (in the presence of 0.8 mM CaCl2 or 40 μM NS1619) conditions. An example of the effect of increasing concentrations of IbTx on NS1619-induced respiration and ΔΨ depolarization is shown in Figure 3A. Under all studied conditions, addition of IbTx up to a concentration of 1.5 μM blocked unstimulated, NS1619-induced, and Ca2+-induced respiration by up to 4%, 53% and 79% respectively (Figure 3C). Similarly, IbTx restored up to 1%, 46% and 78% of unstimulated, NS1619-induced and Ca2+-induced ΔΨ depolarization respectively (Figure 3D). These results indicate low activity of the mitoBKCa channel in the absence of modulators in isolated potato tuber mitochondria, as IbTx has a slight inhibitory effect. Moreover, the results reveal that IbTx inhibits Ca2+-induced mitoBKCa channel activity (~80% inhibition) more efficiently than NS1619-induced channel activity (~50% inhibition), again indicating a non-specific uncoupling effect of NS1619 on isolated potato tuber mitochondria. The relationship between NS1619-induced IbTx-sensitive respiration and various IbTx concentrations reveals that 50% of maximal inhibition (I0.5) by IbTx is reached at 615 nM, as calculated from the linear regression of a double-reciprocal plot (Figure 3B, inset). A similar value was obtained when the effect of increasing IbTx concentration on ΔΨ depolarization was analysed (results not shown).

To exclude an influence of the applied modulators (Ca2+, Ns1619, IbTx) on succinate dehydrogenase activity, experiments were also performed with external NADH as a respiratory substrate (results not shown). The results confirm observations taken from the experiments described above (Figures 1–3).

The findings of the present study strongly suggest the presence of the Ca2+-activated potassium channel in the inner membrane of potato tuber mitochondria. To further test this hypothesis, we investigated the effect of Ca2+ and IbTx on isolated potato tuber mitochondria respiring (with succinate) in incubation media containing different monovalent cations (chloride salts) compared with those incubated in the presence of K+ (Figure 4) or in the absence of any monovalent cations. In medium deprived of monovalent cations, no Ca2+-induced IbTx-sensitive increase in respiration and ΔΨ depolarization were observed (results not shown). Figure 4 shows the influence of increasing concentrations (up to 50 mM) of KCl, NaCl, LiCl, RbCl and CsCl on Ca2+-induced IbTx-sensitive ΔΨ depolarization (i.e. the difference between ΔΨ in the presence of 0.8 mM CaCl2 and ΔΨ in the presence of CaCl2 and 1.5 μM IbTx). The results indicate that the influence of potassium channel modulators (Ca2+ and IbTx) on isolated potato tuber mitochondria in the above experiments can be significantly attributed to K+ influx through the inner membrane.

Cation selectivity: influence of cations on Ca2+-induced IbTx-sensitive resting respiratory rate and membrane potential

Figure 4
Cation selectivity: influence of cations on Ca2+-induced IbTx-sensitive resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium, except that 3 mM NaH2PO4 was replaced with 3 mM Tris/H2PO4. Increasing concentrations of KCl, NaCl, LiCl, CsCl or RbCl (10–50 mM) were obtained by successive additions once a steady-state ΔΨ had been established. The dilution effect of salt addition was taken into account. The change in ΔΨ induced by a given chloride salt relative to the initial State 4 ΔΨ (216±1 mV, ±S.D.) is shown as the difference between ΔΨ measured in the presence of 0.8 mM CaCl2 and ΔΨ measured in the presence of CaCl2 and 1.5 μM IbTx.

Figure 4
Cation selectivity: influence of cations on Ca2+-induced IbTx-sensitive resting respiratory rate and membrane potential

Mitochondria were incubated in a standard incubation medium, except that 3 mM NaH2PO4 was replaced with 3 mM Tris/H2PO4. Increasing concentrations of KCl, NaCl, LiCl, CsCl or RbCl (10–50 mM) were obtained by successive additions once a steady-state ΔΨ had been established. The dilution effect of salt addition was taken into account. The change in ΔΨ induced by a given chloride salt relative to the initial State 4 ΔΨ (216±1 mV, ±S.D.) is shown as the difference between ΔΨ measured in the presence of 0.8 mM CaCl2 and ΔΨ measured in the presence of CaCl2 and 1.5 μM IbTx.

Electrophysiological properties of mitoBKCa channel from the potato tuber mitochondrial membrane

The inner-mitochondrial-membrane-enriched SMPs from potato tuber mitochondria were reconstituted into PLMs. Incorporation of channels into the bilayer was usually observed within 10–20 min after SMP addition to the trans compartment. SMP fractions from five different preparations were used. More than 140 reconstitutions were monitored in the single-channel experiments. Approx. 40% of the channels incorporated into bilayers showed properties of the large-conductance potassium channels. Figure 5 shows representative current–time traces (Figure 5B) and current–voltage relationship (Figure 5C) for potassium channel opening at different voltages in the 50/450 mM KCl (cis/trans) gradient solutions. The current was measured as a function of applied potential at intervals ranging from +90 mV to −30 mV (Figure 5C). The calculated ion conductance is equal to 502±15 pS for potential from −30 to 20 mV and 615±12 pS for potential from +40 to 90 mV, thus indicating large-conductance potassium channel activity. The reversal potential of +34 mV calculated from curve fitting to the experimental data indicates that the examined ion channel is cation-selective. All incorporated large-conductance potassium channels were more active at positive than at 0 mV potentials (Figure 5D). Sometimes we recorded spontaneous closing of channels at 0 mV and at negative potentials (Figure 5B). The activity of such channels could be rescued after applying positive potentials, for example 50 mV.

Single channel recordings of large-conductance potassium channels from potato tuber mitochondria in planar lipid bilayers

Figure 5
Single channel recordings of large-conductance potassium channels from potato tuber mitochondria in planar lipid bilayers

(A) Schematic configuration of the cis and trans compartments used in the experiments. Reconstitution of mitochondrial inner membranes into planar lipid bilayers was performed as described in the Experimental section. (B) Single channel current-time recordings in 50/450 mM KCl (cis/trans) gradient solutions at different voltages. ‘’ Indicates current during the closed state of the channel. Each example is representative of eight measurements. (C) Current–voltage (I/V) characteristics of single-channel events in 50/450 mM KCl (cis/trans) gradient solutions (n=12) at different voltages. (D) Probability of channel opening (P) at two different voltages, 0 and 50 mV (means±S.E.M., n=3).

Figure 5
Single channel recordings of large-conductance potassium channels from potato tuber mitochondria in planar lipid bilayers

(A) Schematic configuration of the cis and trans compartments used in the experiments. Reconstitution of mitochondrial inner membranes into planar lipid bilayers was performed as described in the Experimental section. (B) Single channel current-time recordings in 50/450 mM KCl (cis/trans) gradient solutions at different voltages. ‘’ Indicates current during the closed state of the channel. Each example is representative of eight measurements. (C) Current–voltage (I/V) characteristics of single-channel events in 50/450 mM KCl (cis/trans) gradient solutions (n=12) at different voltages. (D) Probability of channel opening (P) at two different voltages, 0 and 50 mV (means±S.E.M., n=3).

Substances known to modulate mammalian mitoBKCa channel activity were used to examine the properties of the K+ ion channel observed in our experiments. Figure 6 demonstrates the effect of 300 μM Ca2+ on the potato tuber mitochondrial large-conductance potassium channels. Figure 6(A) shows single channel recordings in the 50/450 mM KCl (cis/trans) gradient solutions at 0 mV, before and after addition of Ca2+. Ca2+ caused a transition into an open state that indicates the existence of the mitoBKCa channel in the potato tuber mitochondrial inner membrane. As depicted in Figure 6(B), the number of observations of the channel being in an open state changed from 73% to 86%. The probability of channel opening (P) at 0 mV potential increased from 0.49±0.07 to 0.84±0.05 (n=4, P<0.0076, unpaired t test) in the presence of Ca2+ (Figure 6C).

Effects of Ca2+ on the activity of large-conductance potassium channels from potato tuber mitochondria

Figure 6
Effects of Ca2+ on the activity of large-conductance potassium channels from potato tuber mitochondria

(A) Example of the single channel recordings in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV under control conditions and after addition of 300 μM Ca2+ to the cis and trans compartments (n=4). ‘’ Indicates current during the closed state of the channel. (B) Amplitudes measured in the absence and presence of 300 μM Ca2+. All points shown were fitted by two Gaussian distributions. The closed state corresponds to the peak at 0 pA. O, open state; C, closed state. (C) Probability of channel opening (P) at 0 mV potential. The mean for four different experiments (±S.E.M.) is shown.

Figure 6
Effects of Ca2+ on the activity of large-conductance potassium channels from potato tuber mitochondria

(A) Example of the single channel recordings in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV under control conditions and after addition of 300 μM Ca2+ to the cis and trans compartments (n=4). ‘’ Indicates current during the closed state of the channel. (B) Amplitudes measured in the absence and presence of 300 μM Ca2+. All points shown were fitted by two Gaussian distributions. The closed state corresponds to the peak at 0 pA. O, open state; C, closed state. (C) Probability of channel opening (P) at 0 mV potential. The mean for four different experiments (±S.E.M.) is shown.

Figure 7 presents the inhibitory effect of IbTx on the potato tuber mitochondrial large-conductance potassium channels. As shown in the representative single channel recording obtained in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV, before and after the addition of 600 nM IbTx, the inhibitor caused a complete transition of the channel protein into a closed state (Figure 7A). The number of open-state events changed from 83% to zero after IbTx addition, as illustrated in the histograms (Figure 7B). The probability of channel opening (P) at 0 mV potential decreased from 0.75±0.27 for control recordings to 0.049±0.04 after addition of 600 nM IbTx (P<0.008, unpaired t test). Moreover, IbTx blocked the channel activity in a dose-dependent manner (in a range from 200 to 600 nM) (Figure 7C). The apparent concentration of IbTx that provided half-maximal inhibition (I0.5) was approx. 170 nM. In our PLM experiments, 25% of the channels with a large conductance did not display sensitivity to IbTx (results not shown).

Effects of IbTx on the activity of large-conductance potassium channels from potato tuber mitochondria

Figure 7
Effects of IbTx on the activity of large-conductance potassium channels from potato tuber mitochondria

(A) Example of single channel recordings in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV under control conditions and after addition of 600 nM IbTx to the cis and trans compartments. ‘’ Indicates current during the closed state of the channel. (B) Amplitudes from a control experiment and after addition of 600 nM IbTx. All points shown were fitted by two Gaussian distributions. The closed state corresponds to the peak at 0 pA. O, open state; C, closed state. (C) Probability of channel opening (P) at 0 mV potential. Results are representative of eight experiments in which different concentrations of IbTx were used and are presented as means±S.E.M.

Figure 7
Effects of IbTx on the activity of large-conductance potassium channels from potato tuber mitochondria

(A) Example of single channel recordings in 50/450 mM KCl (cis/trans) gradient solutions at 0 mV under control conditions and after addition of 600 nM IbTx to the cis and trans compartments. ‘’ Indicates current during the closed state of the channel. (B) Amplitudes from a control experiment and after addition of 600 nM IbTx. All points shown were fitted by two Gaussian distributions. The closed state corresponds to the peak at 0 pA. O, open state; C, closed state. (C) Probability of channel opening (P) at 0 mV potential. Results are representative of eight experiments in which different concentrations of IbTx were used and are presented as means±S.E.M.

Immunological detection of plant mitoBKCa channel proteins

Immunoblotting of total mitochondrial proteins, as well as SMPs, allowed immunological detection of the plant mitoBKCa channel. For this, antibodies raised against the mammalian plasma membrane BKCa channel pore (α subunit KCa1.1) were used. In plant mitochondrial and SMP fractions, a protein band with a molecular mass of approx. 70 kDa was detected using the anti-KCa1.1 antibodies (Figure 8A). We also used antibodies raised against the mammalian plasma membrane BKCa channel β subunits. In potato tuber mitochondrial and SMP fractions, anti-sloβ2 antibodies cross-reacted with a single band at approx. 30 kDa (Figure 8B). Less sensitive reactivity was observed with anti-sloβ1 and anti-sloβ4 antibodies (results not shown). As shown in Figure 8, much stronger signals were obtained with SMPs compared with isolated mitochondria using anti-KCa1.1 and anti-sloβ2 antibodies, proving that the detected proteins localized to the inner membrane of potato tuber mitochondria. Moreover, specific blocking peptides blocked the antibody–antigen interaction, demonstrating the specificity of the reaction in the Western blot analysis. On the other hand, mitochondrial (isolated mitochondria and SMPs) and plasma membranes probed with antibodies to a plant plasma membrane marker (H+-ATPase) displayed no signal in the mitochondrial fractions, indicating the absence of surface membrane contamination (Figure 8C). Therefore, we can conclude that the potato tuber mitoBKCa channel may contain subunits similar to the α subunit KCa1.1 and the β subunit sloβ2.

Western blot analysis of potato tuber fractions

Figure 8
Western blot analysis of potato tuber fractions

(A,B) Western blots with anti-KCa1.1 (A) and anti-sloβ2 (B) antibodies raised against the mammalian BKCa channel proteins in the absence or presence of a specific blocking peptide. (C) Detection of H+-ATPase. Mito, mitochondria; PM, plasma membrane-enriched fraction. Different amounts of protein were loaded into each lane (as indicated). Examples of three or four immunoblots (using samples from different preparations) are shown.

Figure 8
Western blot analysis of potato tuber fractions

(A,B) Western blots with anti-KCa1.1 (A) and anti-sloβ2 (B) antibodies raised against the mammalian BKCa channel proteins in the absence or presence of a specific blocking peptide. (C) Detection of H+-ATPase. Mito, mitochondria; PM, plasma membrane-enriched fraction. Different amounts of protein were loaded into each lane (as indicated). Examples of three or four immunoblots (using samples from different preparations) are shown.

DISCUSSION

In the present study, we describe for the first time (to our knowledge) the functional properties of the mitoBKCa channel-like protein in plant mitochondria. Potassium channel activators, Ca2+ and NS1619, are able to modulate the resting respiratory rate (stimulation) and ΔΨ (depolarization) in isolated potato tuber mitochondria. The opposite responses (respiratory rate inhibition and ΔΨ repolarization) were observed when the mitoBKCa channel inhibitor IbTx was applied. These effects were dependent on the presence of K+ in the incubation medium. Thus, we provide evidence that the observed effects of the mitoBKCa channel openers are due to activation of electrogenic potassium transport through the mitochondrial inner membrane, probably mediated by a potassium channel belonging to the family of mitochondrial potassium channels previously described in mammalian mitochondria [7,12].

This is the first study to investigate the electrophysiological properties of the large-conductance potassium channels in plant mitochondria. We used the planar lipid bilayers technique to confirm that large potassium channels are present in the potato tuber mitochondrial inner membrane. The cation selectivity, large conductance (502–615 pS), reverse potential (+34 mV) and sensitivity to Ca2+ and IbTx of the potato tuber mitoBKCa channel indicate similarity to the mammalian mitoBKCa channels previously reported in glioma [8], skeletal muscle [29], brain [30] and cardiac [31] mitochondria. However, the ion conductance of the potato tuber mitoBKCa channel (502–615 pS) is higher than that observed for mammalian mitoBKCa channels (~260–300 pS). This could be due to highly active K+ transport in some plant mitochondria, leading to enormous mitochondrial depolarization (also in potato tuber mitochondria) [14], which contrasts with mammalian mitochondria in which ΔΨ dissipation seems to be weaker [32,33]. Moreover, the mammalian plasma membrane BKCa channel has a higher relative permeability for Rb+ than for Na+ [34] in contrast with that observed for potato tuber mitoBKCa channel. However, K+ flux is dominant.

In addition to the functional studies, the presence of mitoBKCa channel proteins in potato tuber mitochondria is indicated by their cross-reactivity with antibodies raised against the KCa1.1 α subunit of the mouse BKCa channel and the β-subunit of the human sloβ2 BKCa channel. This suggests that the mitoBKCa channel present in the potato tuber mitochondrial inner membrane may be structurally similar to the mammalian BKCa channel. Namely, it may be formed by the principle pore-forming α-subunit that interacts with an auxiliary β2 subunit. The predominant β2-subunit may determine the channel's activity, including its sensitivity to Ca2+ and other modulators. The molecular mass of the detected potato tuber proteins (approx. 70 and 30 kDa for the α- and β2-subunits respectively) differs from that of the mammalian proteins from plasma membrane (~125 and ~44 kDa for the α and β2 subunits respectively) [35], whereas the molecular mass of the α-subunit in mammalian mitochondria is variable, depending on the tissue (55–125 kDa) [31,36]. The β2-subunit of mitoBKCa was detected in astrocyte mitochondria [37]. However, the molecular identity (gene and protein sequences) of the mammalian mitoBKCa channel is presently unknown. On the other hand, the molecular mass of the detected potato tuber mitoBKCa channel α subunit (~70 kDa) is similar to the plant potassium channel protein from the plasma membrane of Arabidopsis thaliana (76 kDa) [38].

Our results in the present study demonstrate the presence of a mitoBKCa channel in plant mitochondria that may function as a possible novel signalling link between intramitochondrial calcium levels and the mitochondrial membrane potential. Ion channels in the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to plant cell function. At least one type of potassium channel, the mitoKATP channel, has been described in plant mitochondria to date [1417]. The physiological functions of the plant mitoKATP channel remain unclear. It may be involved in regulating mitochondrial volume [15], in programmed cell death [19] and/or in the prevention of reactive-oxygen-species formation [14,20]. The physiological role of the mitoBKCa channel in the plant mitochondria described in the present work, which seems to significantly modulate K+ mitochondrial distribution, awaits exploration.

In summary, in the present study we identified and characterized a novel potassium channel of the potato tuber mitochondrial inner membrane. Pharmacological, biophysical and molecular properties of the potato tuber mitochondrial potassium channel are similar to those of the well-known mammalian plasma membrane BKCa, including: (i) potassium selectivity and activation by Ca2+ and NS1619 (a BKCa opener); (ii) inhibition by IbTx (a BKCa blocker); (iii) biophysical properties of a large-conductance potassium channel; and (iv) immunoreactivity with antibodies raised against α- and β-subunits of the plasma membrane BKCa. These properties strongly suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in plant mitochondria. The functional and immunoblotting data obtained in the present study demonstrate for the first time the presence of a mitoBKCa channel in the inner membrane of plant mitochondria.

Abbreviations

     
  • AOX

    alternative oxidase

  •  
  • BKCa channel

    plasma membrane large-conductance Ca2+-activated potassium channel

  •  
  • IbTx

    iberiotoxin

  •  
  • mitoBKCa channel

    mitochondrial large-conductance Ca2+-activated potassium channel

  •  
  • mitoKATP channel

    mitochondrial ATP-sensitive potassium channel

  •  
  • NS1619

    1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one

  •  
  • PLM

    planar lipid membrane

  •  
  • SMP

    submitochondrial particle

  •  
  • TPP+

    tetraphenylphosphonium

  •  
  • ΔΨ

    mitochondrial transmembrane electrical potential

AUTHOR CONTRIBUTION

Izabela Koszela-Piotrowska performed the electrophysiological experiments, analysed and interpreted the data and co-wrote the manuscript. Karolina Matkovic performed the bioenergetic experiments (oxygen uptake and membrane potential measurements) and the immunological analysis. Adam Szewczyk provided scientific guidance and edited the manuscript prior to submission. Wieslawa Jarmuszkiewicz provided scientific expertise and co-wrote the manuscript.

FUNDING

This work was supported by the Polish Mitochondrial Network MitoNet.pl and by the Nencki Institute of Experimental Biology, and was also partially supported by the Ministry of Science and Higher Education [grant numbers P-N/031/2006 and 3382/B/P01/2007/33].

References

References
1
O'Rourke
B.
Evidence for mitochondrial K+ channels and their role in cardioprotection
Circ. Res.
2004
, vol. 
94
 (pg. 
420
-
432
)
2
Szewczyk
A.
Jarmuszkiewicz
W.
Kunz
W. S.
Mitochondrial potassium channels
IUBMB Life
2009
, vol. 
61
 (pg. 
134
-
143
)
3
Szewczyk
A.
The intracellular potassium and chloride channels: properties, pharmacology and function
Mol. Membr. Biol.
1998
, vol. 
15
 (pg. 
49
-
58
)
4
Bernardi
P.
Mitochondrial transport of cations: channels, exchangers, and permeability transition
Physiol. Rev.
1999
, vol. 
79
 (pg. 
1127
-
1155
)
5
Szewczyk
A.
Wojtczak
L.
Mitochondria as a pharmacological target
Pharmacol. Rev.
2002
, vol. 
54
 (pg. 
101
-
127
)
6
Halestrap
A. P.
Regulation of mitochondrial metabolism through changes in matrix volume
Biochem. Soc. Trans.
1994
, vol. 
22
 (pg. 
522
-
529
)
7
Inoue
I.
Nagase
H.
Kishi
K.
Higuti
T.
ATP-sensitive K+ channel in the mitochondrial inner membrane
Nature
1991
, vol. 
352
 (pg. 
244
-
247
)
8
Siemen
D.
Loupatatzis
C.
Borecky
J.
Gulbins
E.
Lang
F.
Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line
Biochem. Biophys. Res. Commun.
1999
, vol. 
257
 (pg. 
549
-
554
)
9
Szabo
I.
Bock
J.
Jekle
A.
Soddemann
M.
Adams
C.
Lang
F.
Zoratti
M.
Gulbins
E.
A novel potassium channel in lymphocyte mitochondria
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
12790
-
12798
)
10
Jarmuszkiewicz
W.
Sluse-Goffart
C. M.
Vercesi
A. E.
Sluse
F. E.
Alternative oxidase and uncoupling protein: thermogenesis versus cell energy balance
Biosci. Rep.
2001
, vol. 
21
 (pg. 
213
-
222
)
11
Sluse
F. E.
Jarmuszkiewicz
W.
Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role
Braz. J. Med. Biol. Res.
1998
, vol. 
31
 (pg. 
733
-
747
)
12
Siedow
J. N.
Umbach
A. L.
The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity
Biochim. Biophys. Acta
2000
, vol. 
1459
 (pg. 
432
-
439
)
13
Vercesi
A. E.
Borecky
J.
Maia
I. D.
Arruda
P.
Cuccovia
I. M.
Chaimovich
H.
Plant uncoupling mitochondrial proteins
Annu. Rev. Plant Biol.
2006
, vol. 
57
 (pg. 
383
-
404
)
14
Pastore
D.
Stoppelli
M. C.
Di Fronzo
N.
Passarella
S.
The existence of the K+ channel in plant mitochondria
J. Biol. Chem.
1999
, vol. 
274
 (pg. 
26683
-
26690
)
15
Petrussa
E.
Casolo
V.
Braidot
E.
Chiandussi
E.
Macri
F.
Vianello
A.
Cyclosporin A induces the opening of a potassium-selective channel in higher plant mitochondria
J. Bioenerg. Biomembr.
2001
, vol. 
33
 (pg. 
107
-
117
)
16
Chiandussi
E.
Petrussa
E.
Macri
F.
Vianello
A.
Modulation of a plant mitochondrial K+ATP channel and its involvement in cytochrome c release
J. Bioenerg. Biomembr.
2002
, vol. 
34
 (pg. 
177
-
184
)
17
Diolez
P.
Moreau
F.
Correlations between ATP-synthesis, membrane potential and oxidation rate in plant mitochondria
Biochim. Biophys. Acta
1985
, vol. 
806
 (pg. 
56
-
63
)
18
Pastore
D.
Trono
D.
Laus
M. N.
Di Fronzo
N.
Flagella
Z.
Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria
J. Exp. Bot.
2007
, vol. 
58
 (pg. 
195
-
210.
)
19
Casolo
V.
Petrussa
E.
Krajnáková
J.
Macrì
F.
Vianello
A.
Involvement of the mitochondrial K+ATP channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures
J. Exp. Bot.
2005
, vol. 
56
 (pg. 
997
-
1006
)
20
Ruy
F.
Vercesi
A. E.
Andrade
P. B.
Bianconi
M. L.
Chaimovich
H.
Kowaltowski
A. J.
A highly active ATP-insensitive K+ import pathway in plant mitochondria
J. Bioenerg. Biomembr.
2004
, vol. 
36
 (pg. 
195
-
202
)
21
Bednarczyk
P.
Kicinska
A.
Kominkova
V.
Ondrias
K.
Dołowy
K.
Szewczyk
A.
Quinine inhibits mitochondrial ATP-regulated potassium channel from bovine heart
J. Membr. Biol.
2004
, vol. 
199
 (pg. 
63
-
72
)
22
Hordejuk
R.
Lobanov
N. A.
Kicinska
A.
Szewczyk
A.
Dolowy
K.
pH modulation of large conductance potassium channel from adrenal chromaffin granules
Mol. Membr. Biol.
2004
, vol. 
21
 (pg. 
307
-
313
)
23
Bednarczyk
P.
Dołowy
K.
Szewczyk
A.
Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart
FEBS Lett.
2005
, vol. 
579
 (pg. 
1625
-
1632
)
24
Kamo
N.
Muratsugu
M.
Hongoh
R.
Kobatake
Y.
Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state
J. Membr. Biol.
1979
, vol. 
49
 (pg. 
105
-
121
)
25
Silva
M. A. P.
Carnieri
E. G. S.
Vercesi
A. E.
Calcium transport by corn mitochondria: evaluation of the role of phosphate
Plant Physiol.
1992
, vol. 
98
 (pg. 
452
-
457
)
26
de Oliveira
H. C.
Saviani
E. E.
de Oliveira
J. F. P.
Salgado
I.
Cyclosporin A inhibits calcium uptake by Citrus sinensis mitochondria
Plant Sci.
2007
, vol. 
172
 (pg. 
665
-
670
)
27
Heinen
A.
Camara
A. K. S.
Aldakkak
M.
Rhodes
S. S.
Riess
M. L.
Stowe
D. F.
Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential
Am. J. Physiol. Cell Physiol.
2006
, vol. 
292
 (pg. 
C148
-
C156
)
28
Heinen
A.
Winning
A.
Shlack
W.
Hellmann
M. W.
Precel
B.
Fraβdorf
J.
Weber
N. C.
Physiological levels of glutamine prevent morphine-induced preconditioning in the isolated rat heart
Eur. J. Pharmacol.
2008
, vol. 
578
 (pg. 
108
-
113
)
29
Skalska
J.
Piwonska
M.
Wyroba
E.
Surmacz
L.
Wieczorek
R.
Koszela-Piotrowska
I.
Zielinska
J.
Bednarczyk
P.
Dolowy
K.
Wilczyñski
G. M.
, et al. 
A novel potassium channel in skeletal muscle mitochondria
Biochim. Biophys. Acta
2008
, vol. 
1777
 (pg. 
651
-
659
)
30
Skalska
J.
Bednarczyk
P.
Piwonska
M.
Kulawiak
B.
Wilczynski
G.
Dolowy
K.
Kudin
A. P.
Kunz
W. S.
Szewczyk
A.
Calcium ions regulate K uptake into brain mitochondria: the evidence for a novel potassium channel
Int. J. Mol. Sci.
2009
, vol. 
10
 (pg. 
1104
-
1120
)
31
Xu
W.
Liu
Y.
Wang
S.
McDonald
T.
Van Eyk
J. E.
Sidor
A.
O'Rourke
B.
Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane
Science
2002
, vol. 
298
 (pg. 
1029
-
1033
)
32
Debska
G.
Kicinska
A.
Skalska
J.
Szewczyk
A.
May
R.
Elger
C. E.
Kunz
W. S.
Opening of potassium channels modulates mitochondrial function in rat skeletal muscle
Biochim. Biophys. Acta
2002
, vol. 
1556
 (pg. 
97
-
105
)
33
Debska
G.
May
R.
Kiciñska
A.
Szewczyk
A.
Elger
C. E.
Kunz
W. S.
Potassium channel openers depolarize hippocampal mitochondria
Brain Res.
2001
, vol. 
892
 (pg. 
42
-
50
)
34
Blatz
A. L.
Magleby
K. L.
Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle
J. Gen. Physiol.
1984
, vol. 
84
 (pg. 
1
-
23
)
35
Wulf
H.
Hay-Schmidt
A.
Poulsen
A. N.
Klaerke
D. A.
Olesen
J.
Jansen-Olesen
I.
Molecular investigations of BKCa channels and the modulatory beta-subunits in porcine basilar and middle cerebral arteries
J. Mol. Histol.
2009
, vol. 
40
 (pg. 
87
-
97
)
36
Douglas
R. M.
Lai
J. C. K.
Bian
S.
Cummins
L.
Moczydlowski
E.
Haddad
G. G.
The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain
Neuroscience
2006
, vol. 
139
 (pg. 
1249
-
1261
)
37
Piwonska
M.
Wilczek
E.
Szewczyk
A.
Wilczyñski
G. M.
Diferential distribution of Ca2+-activated channel β4 subunit in rat brain: immunolocalization in neuronal mitochondria
Neuroscience
2008
, vol. 
153
 (pg. 
446
-
60
)
38
Reintanz
B.
Szyroki
A.
Ivashikina
N.
Ache
P.
Godde
M.
Becker
D.
Palme
K.
Hedrich
R.
AtKC1, a silent Arabidopsis potassium channel α-subunit modulates root hair K+ influx
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
4079
-
4084
)

Author notes

1

These authors contributed equally to this work.