Experimental Neurology 212 (2008) 543–547
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Experimental Neurology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex n r
BK channel openers inhibit ROS production of isolated rat brain mitochondria
Bogusz Kulawiak a, Alexei P. Kudin b,c, Adam Szewczyk a, Wolfram S. Kunz b,c,⁎
a
b
c
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland
Department of Epileptology, University Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany
Life and Brain Center, University Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany
A R T I C L E
I N F O
Article history:
Received 20 December 2007
Revised 23 April 2008
Accepted 10 May 2008
Available online 20 May 2008
Keywords:
BK channel opener
Brain mitochondria
ROS production
A B S T R A C T
To delineate the potential mechanism of neuroprotective effects of potassium channel openers we have
investigated, how Ca2+-activated large conductance potassium channel (BKCa channel) openers influence the
production of reactive oxygen species (ROS) by rat brain mitochondria, since mitochondrial generation of
ROS is known to have a crucial influence on neuronal survival. We studied the effects of BKCa channel openers
CGS 7184 and NS 1619 on hydrogen peroxide production rate of isolated rat brain mitochondria. In K+containing media 3 μM of both channel openers reduced the hydrogen peroxide production rates by
approximately 20%. This effect was not observed in Na+-containing media. This potassium-dependent partial
inhibition of hydrogen peroxide production was found to be sensitive to the selective blockers of BKCa
channel iberiotoxin and charybdotoxin applied in nanomolar concentrations. Taken together, our data are
compatible with the viewpoint that the opening of a Ca2+-activated large conductance potassium channel
being localised in the inner membrane of brain mitochondria inhibits ROS production by respiratory chain
complex I. This finding is suggested to explain the beneficial effects of BK potassium channel openers on
neuronal survival.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Due to their putative protective role in a large number of cellular
pathologies potassium channels (K+ channels) are extensively studied
(Facundo et al., 2006; Ohya et al., 2005; Szewczyk et al., 2006). Similar
to openers of KATP channels (Kowaltowski et al., 2006; Mattson and
Liu, 2003; Yang et al., 2004) also certain openers of Ca2+-activated
large conductance potassium channels (BK channels) appear to rescue
neurons affected by different insults (Cheney et al., 2001; RundenPran et al., 2002). The beneficial effects of opening of BK channels on
neuronal survival have been attributed primary to plasma membrane
hyperpolarization, since BK channels are voltage- and calciumdependent potassium channels whose activation tends to reduce
cellular excitability (Runden-Pran et al., 2002). But additionally to the
well established effects on plasma membrane channels also potential
mitochondrial targets of BK channel openers and blockers have been
described. Thus, a putative mitochondrial large conductance Ca2+activated potassium channel (mitoBKCa channel) has been described
in human glioma cells LN229 using patch-clamp technique (Siemen
et al., 1999). This channel, with a conductance of 295 pS, was
stimulated by Ca2+ and blocked by charybdotoxin. Later, the presence
of a channel with properties similar to the plasma membrane BK
⁎ Corresponding author. Department of Epileptology, University Bonn Medical
Center, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany.
E-mail address: [email protected] (W.S. Kunz).
0014-4886/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2008.05.004
channel (stimulated by the potassium channel opener NS 1619 and
blocked by charybdotoxin, iberiotoxin and paxilline) was observed in
cardiac mitochondria (Ohya et al., 2005; Sato et al., 2005; Xu et al.,
2002). This potassium channel has been proposed to affect mitochondrial metabolism due to regulation of matrix volume. In addition to
this physiological function, a pivotal role of mitochondrial potassium
channels has been implicated in cardio- and neuroprotection. The key
effect of potassium channel activation associated with cytoprotection
appears to be the modulation of the generation rate of mitochondrial
ROS (reactive oxygen species) (Andrukhiv et al., 2006; Facundo et al.,
2007; Heinen et al., 2007a). In previous studies on heart mitochondria,
it remained unclear whether ROS production increased (Andrukhiv
et al., 2006; Facundo et al., 2006) or decreased (Facundo et al., 2007;
Heinen et al., 2007b) upon activation of potassium transport. To
resolve this discrepancy and to extend these investigations to brain
tissue, where also cytoprotective effects of BK channel openers have
been described (Cheney et al., 2001; Runden-Pran et al., 2002), we
studied the effects of openers of the large conductance Ca2+-activated
potassium channel on the hydrogen peroxide production of isolated
rat brain mitochondria (Kudin et al., 2004).
Materials and methods
All standard chemicals were purchased from Sigma-Aldrich
(Taufkirchen). Iberiotoxin and charybdotoxin were obtained from
Alomone Labs (Jerusalem). The uncoupler TTFB (4,5,6,7-tetrachloro-2trifluoromethylbezimidazole) is a kind gift from Prof. B. Beechey
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B. Kulawiak et al. / Experimental Neurology 212 (2008) 543–547
(Aberystwyth) and the potassium channel opener CGS 7184 is a kind
gift from Dr. Michele Chiesi, Novartis Pharma (Basel).
Rat brain mitochondria were isolated according to the protocol
described by Rosental et al. (1987) with a small modification which
allowed to obtain mitochondria with much better functional characteristics. In brief, the isolation protocol was the following: before
mitochondrial isolation all solutions were cooled down till the slight
appearance of ice. One Wistar rat (60–90 days old) was anaesthetized
by chloroform and killed by decapitation. Its brain was immediately
transferred into the ice-cold MSE solution (225 mM mannitol, 75 mM
sucrose, 1 mM EGTA, 5 mM HEPES, 1 mg/ml BSA, pH 7.4) and shaken to
wash out blood. Then we minced the brain, added 10 ml of ice-cold
MSE-nagarse solution (0.05% nagarse in MSE solution) and homogenized it at 600 rpm/min using a potter homogenizer. Thereafter, we
added 20 ml ice-cold MSE solution and centrifuged the homogenate at
2000 ×g for 4 min. After centrifugation we passed the supernatant
through a cheesecloth and centrifuged it at 12000 ×g for 9 min. To
permeabilize synaptosomes we dissolved the resulting pellet in 10 ml
ice-cold MSE-digitonin solution (0.02% digitonin in MSE solution),
transferred the solution to a small glass homogenizer and homogenized it 8–10 times manually to obtain a homogenous suspension.
Finally, we centrifuged the suspension at 12000 ×g for 11 min and
dissolved the resulting pellet in about 300 μl MSE solution to obtain
about 20 mg protein/ml. The quality of mitochondria was assessed by
determinations of oxygen consumption at 30 °C in MTP medium
(10 mM KH2PO4, 60 mM KCl, 60 mM Tris–HCl, 110 mM mannitol,
0.5 mM EDTA (pH 7.4)) with a PC-supported Oroboros high resolution
oxygraph (Kudin et al., 2004). The respiratory control coefficients with
10 mM glutamate and 5 mM malate as substrates were routinely
higher than 6. The protein content of mitochondria was determined
using a protein assay kit based on Peterson's modification of the
micro-Lowry method according to the instructions of the manufacturer (Sigma-Aldrich).
Mitochondrial H2O2 generation was measured at 30 °C in oxygensaturated MTP medium by monitoring the change in fluorescence of
200 μM p-hydroxyphenylacetic acid (λex = 317 nm, λem = 414 nm)
catalyzed by 20 U/ml horseradish peroxidase in the presence of 15 U
superoxide dismutase (SOD) (Liu et al., 2002). The excess SOD was
added to enable complete conversion of all produced superoxide into
hydrogen peroxide (cf. Kudin et al., 2005). The fluorescence signal was
calibrated by addition of hydrogen peroxide (using 6 additions of
140 pmol H2O2) in the presence of p-hydroxyphenylacetic acid, SOD,
horseradish peroxidase and 0.2 mg/ml of mitochondria. The sodiumcontaining MTP medium consisted of 10 mM NaH2PO4, 60 mM NaCl,
60 mM Tris–HCl, 110 mM mannitol, 0.5 mM EDTA (pH 7.4).
The mitochondrial NAD(P)H fluorescence was measured at 30 °C in
oxygen-saturated MTP medium with a Shimadzu RF5001 spectrofluorimeter at λex = 340 nm and λem = 450 nm.
All data are presented as means ± SD and p values smaller than 0.05
(according to two-sided t-test) were considered to be statistically
significant.
Results
Fig. 1 shows the measurement of generation of hydrogen peroxide by isolated rat brain mitochondria using the fluorescent probe
p-hydroxyphenylacetate. The addition of the respiratory chain substrate succinate to a suspension of intact rat brain mitochondria
resulted in a substantial increase of fluorescence in time, indicating the
production of hydrogen peroxide. This rate of hydrogen peroxide
production was in potassium-containing MTP medium lowered by
addition of 3 μM potassium channel opener CGS 7184 (Hu et al., 1997)
(Fig. 1A, see the downward deflection of the fluorescence trace from
the dashed line). In MTP medium with all potassium ions replaced by
sodium ions similar rates of hydrogen peroxide generation were
obtained, but the addition of 3 μM CGS 7184 was without effect (Fig. 1B,
Fig. 1. Representative experimental traces showing the effect of the potassium channel
opener CGS 7184 (Hu et al., 1997) on H2O2 generation by isolated rat brain
mitochondria. The generation of H2O2 by mitochondria (0.22 mg protein/ml) was
measured fluorimetrically in the presence of 10 mM succinate following the oxidation
of 200 μM p-hydroxyphenylacetic acid in oxygen-saturated medium in the presence of
horseradish peroxidase (20 U/ml). Additionally, an excess of superoxide dismutase
(15 U/ml) was present during the experiment. A — 3 μM CGS 7184 caused decrease of
H2O2 production in potassium-containing medium, B — the effect of CGS 7184 was
absent in sodium-containing medium, C — in the presence of BKCa channel inhibitor
iberiotoxin (IbTx, 50 nM) the effect of CGS 7184 was not observed.
no deflection of the fluorescence trace from the dashed line). This
indicates that the presence of potassium ions is required to alter the
hydrogen peroxide production rate by this potassium channel opener.
To test if the potassium-dependent inhibition of hydrogen peroxide
production is related to a specific potassium channel we applied the
selective blocker of the BKCa channel iberiotoxin (Fig. 1C). Very clearly,
the CGS 7184 addition was in the presence of both, potassium ions and
50 nM iberiotoxin, almost without effect on the hydrogen peroxide
generation rate (no deflection of the fluorescence trace from the dashed
line). To test the influence of the BKCa channel blocker charybdotoxin we
performed separate parallel experiments in potassium medium. We
observed in the presence of 5 μM CGS 7184 alone 81.5%± 6.5% of the
control hydrogen peroxide production, which increased in the additional presence of 250 nM charybdotoxin to 93.3% ± 4.5% (4 separate
mitochondrial preparations). Both, iberiotoxin and charybdotoxin had at
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545
Fig. 3. Experimental traces showing the effect of the potassium channel opener CGS 7184
on NAD(P)H fluorescence of isolated rat brain mitochondria. Mitochondrial protein 0.2 mg/
ml, other experimental conditions like in the legend to Fig. 1. A — 5 μM CGS 7184 caused
decrease of NAD(P)H fluorescence in potassium-containing medium, B — the effect of CGS
7184 was almost absent in sodium-containing medium, C — in the presence of BKCa channel
inhibitor iberiotoxin (IbTx, 50 nM) the effect of CGS 7184 was very small. The addition of
1 μM of the uncoupler TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbezimidazole) caused
under all conditions the complete oxidation of NAD(P)H. The fluorescence scale (ΔF) is
expressed in arbitrary units.
Fig. 2. A: Dose dependence of effects of CGS 7184 on H2O2 production rate by rat brain
mitochondria in the presence of 10 mM succinate in potassium-containing medium
(filled circles) and in sodium-containing medium (open circles). Experimental
conditions like in the legend to Fig. 1. The ROS production is expressed in percent of
initial H2O2 production rate. B: The potassium channel opener NS 1619 inhibits H2O2
production by brain mitochondria in the presence of 10 mM succinate in potassiumcontaining medium (filled circles) and in sodium-containing medium (open circles) in a
dose dependent manner. C: Effects of small concentrations of the uncoupler TTFB
(4,5,6,7-tetrachloro-2-trifluoromethylbezimidazole) on H2O2 production by brain
mitochondria in the presence of 10 mM succinate in potassium-containing medium
(filled circles) and in sodium-containing medium (open circles). The hydrogen peroxide
production rate is expressed in the percent of initial H2O2 production rate. The average
initial H2O2 production rate in potassium-containing medium was 1.4 ± 0.4 nmol/min/
mg protein and in sodium-containing medium 2.1 ± 0.6 nmol/min/mg protein. Number
of independent titration experiments with separate mitochondrial preparations: 4.
the applied concentrations no effects on the hydrogen peroxide production rate, which was in potassium medium with succinate alone 1.28±
0.18 nmol H2O2/min/mg protein, in the additional presence of 250 nM
charybdotoxin 1.23± 0.21 nmol H2O2/min/mg protein or in the additional
presence of 50 nM iberiotoxin 1.26± 0.35 nmol H2O2/min/mg protein (4
separate mitochondrial preparations).
In Fig. 2A the concentration dependency of the CGS 7184 effect on
the hydrogen peroxide generation by rat brain mitochondria is shown.
Approximately 1 μM of this potassium channel opener is required to
cause an about 20% inhibition of hydrogen peroxide production, which
was almost completely potassium-specific (compare filled and open
circles). Similar to this compound, also the potassium channel opener
NS 1619 caused an about 20% inhibition of hydrogen peroxide generation, but the inhibitory effects of this particular substance were
in comparison to CGS 7184 less potassium-selective (Fig. 2B). In
control experiments we determined the effects of mild uncoupling on
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hydrogen peroxide production from succinate. As shown in Fig. 2C,
small additions of the uncoupler TTFB resulted in an inhibition of H2O2
production which was independent on the ion composition of the
medium (cf. open and closed circles). The half maximal inhibition of
hydrogen peroxide generation was detected at TTFB concentrations,
which were required to obtain half maximal uncoupling of mitochondrial respiration with succinate as substrate (18.6 ± 3.6 nM in
potassium medium and 19.3 ± 4.2 nM in sodium medium, n = 3).
To establish whether the mild inhibitory effects of potassium
channel openers on hydrogen peroxide production are of functional
significance, we determined the influence of CGS 7184 additions on
mitochondrial NAD redox state. As shown in Fig. 3A, 5 μM CGS 7184
caused an approximately 15% decrease of the NAD(P)H fluorescence
signal in the presence of succinate. This is an indication for a partial reoxidation of the mitochondrial NAD system. In experiments performed in sodium medium (Fig. 3B) or in potassium medium in the
presence of 50 nM iberiotoxin (Fig. 3C) the oxidation of NAD(P)H by
CGS 7184 was considerably smaller.
To test whether the inhibitory effects of potassium channel openers
on mitochondrial hydrogen peroxide production might be substratedependent we applied in addition to 10 mM succinate alone (Fig. 4A)
also the more physiological substrate combination 10 mM glutamate
+ 5 mM malate (Fig. 4B). These substrates resulted in approximately 6-
Fig. 4. Inhibitory effect of 5 μM CGS 7184 on H2O2 production by rat brain mitochondria
in the presence of different respiratory substrates: A — 10 mM succinate, B — 10 mM
glutamate + 5 mM malate. The results are expressed as a percentage of initial H2O2
production rate. The average initial hydrogen peroxide production rate in the presence
of succinate was 1.3 ± 0.2 nmol H2O2/min/mg protein and in the presence of glutamate +
malate was 0.24 ± 0.06 nmol H2O2/min/mg protein. Where indicated 50 nM iberiotoxin
(IbTx) were added. Number of independent experiments with separate mitochondrial
preparations: 4.
fold lower overall hydrogen peroxide production rates (cf. legend to
Fig. 4), which is in agreement with the lower NAD(P)H redox state (data
not shown). Nevertheless, as visible in Fig. 4B, we detected no difference
in the relative inhibitory effect of CGS 7184 on the hydrogen peroxide
production rates.
Discussion
In this work we observed inhibitory effects of potassium channel
openers CGS 7184 (Hu et al., 1997) and NS 1619 on the generation of
hydrogen peroxide by isolated brain mitochondria. In some of the
presented experiments we used succinate as substrate which serves
under resting state conditions as a donor of electrons for superoxide
generation by reversed electron flow through respiratory chain
complex I. Under these conditions molecular oxygen is reduced to
superoxide by the FMN moiety of complex I — the site being responsible
for physiological superoxide generation in brain mitochondria (Kudin
et al., 2004; Sazanov and Hinchliffe, 2006). Since this redox site is in
contact to the mitochondrial matrix space, the respiratory chain
produced superoxide is readily converted by Mn-superoxide dismutase
to the membrane permeable hydrogen peroxide.
Our data further indicate that the studied potassium channel openers
do not exclusively affect the reversed electron flow driven hydrogen
peroxide production, which is highly dependent on mitochondrial
membrane potential. We observed with the more physiological relevant
substrate combination glutamate + malate that, in analogy to the
experiments with succinate as substrate, CGS 7184 caused an about
20% inhibition of the hydrogen peroxide generation rate. This effect is
also very likely related to the opener caused dissipation of mitochondrial
membrane potential, since uncoupling reduced the glutamate + malate
caused hydrogen peroxide generation by 55 ± 4% (n = 3).
These findings are in line with the expected bioenergetic effects of
opening of potassium channels in the inner mitochondrial membrane
under resting state conditions of a highly reduced NAD system and of a
high membrane potential. The net flux of potassium ions from the cytosol
into the mitochondrial matrix space would lead to a ‘mild uncoupling’,
resulting in a lowered mitochondrial membrane potential and lowered
redox state of the NAD system causing a diminished production of
reactive oxygen species by respiratory chain complex I. The oxidation of
mitochondrial NAD(P)H by potassium channel openers was directly
confirmed with our NAD(P)H fluorescence measurements. ‘Mild
uncoupling’ as potential mechanism to reduce ROS production has
been proposed in previous work to explain tissue protection against ROS
toxicity (Skulachev, 1996). Therefore, our data suggest that openers of BK
channels could in principle deploy their neuro- and cardioprotective
action by this particular mechanism. It remains, however, to be
elucidated yet whether the small changes in ROS production triggered
by BK channel opening are sufficient to completely explain the neuroand cardioprotective effects of the openers.
Our findings seem to be in contrast to previous work by Andrukhiv
et al. (2006) and Heinen et al. (2007a) who detected in heart
mitochondria an increase of complex I-dependent ROS production by
the potassium channel openers cromakalim (50 μM) or NS 1619
(30 μM), respectively. But more recently, Heinen et al. (2007b) reported
for guinea pig heart mitochondria a strong inhibition of succinatesupported hydrogen peroxide generation by 30 μM NS 1619, which
could be partially released by 5 μM of the BK channel blocker paxilline.
One reason for discrepancies to our data might be the heart specific
effects of these drugs. However, it needs to be mentioned, that these
authors used very high concentrations of the particular openers – they
applied 30 μM NS 1619 – while in our study 3 μM NS 1619 had already
the strongest potassium-selective effect. We observed, that concentrations of NS 1619 above 10 μM considerably inhibited the H2O2
production also in sodium-containing media — already 3 μM NS 1619
caused in sodium medium an about 15% inhibition of hydrogen
peroxide production rate (cf. Fig. 2B, open circles). This is very likely
B. Kulawiak et al. / Experimental Neurology 212 (2008) 543–547
caused by side effects of this compound, since high concentrations of
NS 1619 are inhibiting mitochondrial respiratory chain (Kicinska and
Szewczyk, 2004).
A further aspect of our work is related to the fact that the observed
effects of potassium channel openers in the lower micromolar range
are strictly potassium-dependent. Moreover, they were found to be
sensitive to nanomolar concentrations of the well known highly
specific blockers of the large conductance Ca2+-activated potassium
channel iberiotoxin and charybdotoxin. These findings exclude
unspecific pharmacological side effects of both BK channel openers
(cf. Kicinska and Szewczyk, 2004) as potential cause for the partial
inhibition of ROS production. Therefore, both observations strongly
support the proposed presence of a BKCa channel in the inner membrane of brain mitochondria similar as postulated in patch-clamp
observations of mitochondria from glioma cells (Siemen et al., 1999)
and heart (Xu et al., 2002). Supportive evidence for the potential
presence of a BKCa channel in the inner membrane of brain mitochondria has been also obtained with the use of immunocytochemistry and immuno-gold electron microscopy (Douglas et al., 2006).
Summarising, our data are compatible with the viewpoint that the
opening of a large conductance Ca2+-activated potassium channel in
brain mitochondria inhibits complex I-dependent mitochondrial ROS
production, which is proposed to be highly relevant for the tissue
protective effects of potassium channel openers.
Acknowledgments
This study was supported by grants of the Deutsche Forschungsgemeinschaft (KU-911/15-1, SCHR-562/4-3) and the BMBF (01GZ0704) to
WSK and of the Ministry of Science and Higher Education (P-N/031/
2006) to AS.
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