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FEBS Letters xxx (2010) xxx–xxx
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Review
Mitochondrial potassium channels and reactive oxygen species
Dominika Malinska a, Sandra R. Mirandola b, Wolfram S. Kunz b,*
a
b
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur st., 02-093 Warsaw, Poland
Department of Epileptology and Life & 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 30 November 2009
Revised 5 January 2010
Accepted 11 January 2010
Available online xxxx
Edited by Adam Szewczyk
a b s t r a c t
Pretreatment of tissues with potassium channel openers (KCO’s) has been observed to be cytoprotective in a broad variety of insults. This phenomenon has been proposed to be intimately linked
to activation of mitochondrial potassium channels which apparently modulate the mitochondrial
production of reactive oxygen species (ROS). This critical review summarizes literature findings
about the mitochondrial production of ROS, the action of KCO’s on mitochondrial ROS production
and the putative link to the cytoprotective action of these drugs.
Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Keywords:
Mitochondria
Oxidative stress
Potassium channel
Respiration
Electron transport chain
Uncoupling
1. Introduction. Mitochondrial generation of reactive oxygen
species
Reactive oxygen species (ROS) are highly reactive molecules
originating from molecular oxygen which have been implicated
to be important for a broad variety of pathologies and ageing, as
well as for intracellular signaling [1]. The superoxide anion – the
product of one-electron reduction of oxygen – is the precursor of
most ROS. Its dismutation reaction produces hydrogen peroxide,
which in turn can be split by catalase to water and oxygen or within the Fenton reaction partially reduced to the extremely reactive
hydroxyl radical. Well known cell specific enzymatic sources of
superoxide include NADPH oxidases, cytochrome P450-dependent
oxygenases and xanthine oxidase. In most tissues the primary
source of superoxide are certain redox centers of the mitochondrial
respiratory chain [2], which are able for single electron transfer at
the appropriate redox potential enabling reduction of molecular
oxygen, having a standard redox potential of 160 mV [3]. Several
sites within the mitochondrion have been implicated for donating
Abbreviations: KCO’s, potassium channel openers; ROS, reactive oxygen species;
KATP, ATP-regulated potassium channel; BKCa, calcium-activated potassium channel; IPC, ischemic preconditioning; 5-HD, 5-hydroxydecanoic acid; SDH, succinate
dehydrogenase
* Corresponding author. Address: Division of Neurochemistry, Department of
Epileptology, University Bonn Medical Center, Sigmund-Freud-Str. 25, D-53105
Bonn, Germany.
E-mail address: [email protected] (W.S. Kunz).
these electrons, including FMNH2 of respiratory chain complex I
[4,5], and semiquinone at center ‘o’ of respiratory chain complex
III [6,7], which both are probably the most relevant sites for brain
and muscle tissue [8,9]. Moreover, several mitochondrial flavoproteins, like the a-lipoamide dehydrogenase moiety of the a-ketoglutarate dehydrogenase complex [10], the electron-transfer
flavoprotein of the b-oxidation pathway [11] and a-glycerophosphate dehydrogenase [12] have been also suggested as possible
additional sites for mitochondrial ROS production.
As depicted in Scheme 1, the major sites at complex I and III release the membrane impermeable superoxide anion to different
compartments – the FMNH2 – site of complex I to the mitochondrial matrix space and the semiquinone center ‘o’ of complex III
to the intermembrane space [7,8,11]. While most experimental
data suggest that the complex I-produced superoxide anion remains trapped in the matrix space [8,11], the complex III-produced
superoxide apparently can escape the intermembrane space
trough the voltage gated anion channel of the outer mitochondrial
membrane (VDAC; porin pore) [13]. On the other hand, the dismutation product of superoxide – hydrogen peroxide – can cross the
mitochondrial inner membrane presumably by aquaporin 9 channels [14]. There have been also reports assuming matrix superoxide release via the mitochondrial chloride channel [15] but direct
experimental evidence for efficient superoxide release by this
pathway is so far lacking.
Concerning the velocity of superoxide formation, it is important
to note, that this rate is controlled by two factors: (i) the
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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D. Malinska et al. / FEBS Letters xxx (2010) xxx–xxx
Scheme 1. Localization of major mitochondrial superoxide producing sites at complexes I and III. SOD1, Cu,Zn superoxide dismutase; SOD2, Mn superoxide dismutase;
FMNH2, reduced flavine adenine mononucleotide; SDH, succinate dehydrogenase; VDAC, voltage-dependent anion channel (porin pore); SQo, semiquinone anion at center ‘o’;
SQi, semiquinone anion at center ‘i’; Fe2S2, Rieske iron sulphur center; bH, ‘high potential’ cytochrome b562; bL, ‘low potential’ cytochrome b566; IMM, inner mitochondrial
membrane.
concentration of the electron donors – FMNH2 or SQo – and (ii) the
concentration of oxygen [16]. As shown in Scheme 1, this implies
for the complex I site that processes which enhance the supply
of NADH or diminish its oxidation (inhibition of electron flow
through respiratory chain) would increase the concentration of
FMNH2 thus increasing the rate of superoxide formation. In line
with this concept for complex I-dependent superoxide formation
the unique determinant appears to be the mitochondrial NAD-redox state [9]. An important, pathology relevant process, proposed
to increase complex I-dependent ROS formation, is PTP opening,
leading to inhibition of NADH oxidation due to dissociation of
cytochrome c [17]. This mechanism is particularly important to explain increased ROS formation during reperfusion injury. Since the
redox state of the mitochondrial NAD system is controlled by substrate supply (reducing equivalent influx) and respiratory chain
activity (reducing equivalent efflux), it is clear that stimulation of
respiratory chain electron flow by decrease of mitochondrial membrane potential always would decrease the rate of complex Idependent superoxide formation. The respiratory activity is
strongly activated when uncouplers of the oxidative phosphorylation increase the proton permeability of mitochondrial membrane
and waste the redox energy by means of a futile proton cycle [18].
A physiological process which uses this principle is ‘mild mitochondrial uncoupling’, which has been proposed to ameliorate
the complex I-dependent ROS production by reducing reverse electron flow [18,19]. These changes in mitochondrial energy metabolism are promoted by uncoupling proteins [20] but the redox
energy can be also dissipated by rotenone-insensitive external
and internal NAD(P)H dehydrogenases or the alternative, cyanide-resistant, terminal oxidases (AOX) of plant, fungal and protozoan mitochondria [21,22]. Both mechanisms decrease efficiency
of oxidative phosphorylation and increase electron transport,
resulting in a physiological control of complex I-dependent ROS
formation [23].
On the other hand, the concentration of the semiquinone at center ‘o’ of bc1 complex, which is the direct donor of electrons for
superoxide formation at complex III – SQo – is kinetically controlled
by the electron transfer reaction within the Q-cycle (Scheme 1).
Since the concentration of this relevant semiquinone species is
the highest at intermediate levels of CoQ reduction, this implies –
in contrast to complex I-dependent superoxide production – for
complex III a bell-shaped dependency of superoxide production
rate on the reduction state of the entire coenzyme Q pool [24]. That
explains experimental findings in presence of antimycin that at a
high initial state of CoQ reduction inhibitors of succinate oxidation,
like malonate or TTFA [24], but also protonophores and ionophores
enhanced superoxide formation rates from this particular site [25],
while on the other hand, complete inhibition of succinate dehydrogenase inhibited ROS production [25]. This site is also due to the
limited activity of intermembrane space SOD1 [26] potentially able
to liberate superoxide to the cytosolic space (cf. Scheme 1). Therefore, complex III-dependent ROS formation, which can potentially
be activated under conditions of a highly reduced coenzyme Q pool
by depolarization of mitochondrial inner membrane, but also by
inhibition of succinate dehydrogenase, could be important for the
activation of ROS-dependent signal transduction pathways. For this
reason it is plausible to suggest that this particular site might be involved in the preconditioning effects observed upon application of
potassium channel openers (for details, see Section 3).
2. Effects of potassium channel openers on mitochondrial ROS
production
After the functional identification of potassium channels in the
inner mitochondrial membrane [27] and the demonstration, that
cytoprotective properties of certain potassium channel openers
(KCO’s) apparently result from stimulation of mitochondrial ROS
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generation by these chemicals [28] there grew an interest in the
relation between mitochondrial potassium fluxes and ROS generation by the respiratory chain. The earliest and most intensively
studied mitochondrial potassium channel is the ATP regulated
channel (mitoKATP). Recently, also a mitochondrial large conductance calcium activated potassium channel (mitoBKCa) is gaining
increasing attention [29]. It is widely accepted that activation of
potassium channels in the inner mitochondrial membrane and, in
consequence, increasing potassium influx into the matrix leads to
a decrease in mitochondrial membrane potential and consecutive
stimulation of respiration rate leading to matrix alkalinization
(Scheme 2, [30,31]). In contrary to mitochondrial calcium uptake,
where a decrease in membrane potential is a secondary effect
resulting from intracellular calcium buffering role played by mitochondria, potassium uptake by these organelles can be a convenient mechanism for adjusting the mitochondrial metabolic state
to the needs of the cell. Yet, there is not much agreement on
how mitochondrial potassium fluxes influence mitochondrial
superoxide generation. Studies on heart mitochondria provided
so far conflicting results: ROS production increased [32,33] or decreased [34,35] upon activation of potassium transport by application of various potassium channel openers. Such inconsistency can
result from the complexity of responses of the different mitochon-
Scheme 2. Potential effects of KCO’s on mitochondrial ROS production and there
putative link to cytoprotection. Thick red arrows indicate the expected effects of
KCO’s on the mitochondrial ROS producing sites at complexes I and III via mitoKATP
and mitoBKCa, dashed arrows indicate potential side effects. IMM – inner
mitochondrial membrane, OMM – outer mitochondrial membrane.
Table 1
Synopsis of the expected effects of potassium channel openers on mitochondrial ROS
generation.
Documented
biochemical
action of KCO’s
Expected action on ROS-generating sites
Complex I (ROS
species liberated
to cytosol: hydrogen
peroxide)
Complex III (ROS
species liberated
to cytosol: superoxidec)
Mild uncoupling [31]
Matrix swelling [55]
Inhibition of succinate
dehydrogenaseb [37]
Inhibition of complex I of
respiratory chainb [38]
Inhibition
Inhibition
Inhibition/no effect
Stimulation/no effecta
Stimulation/no effecta
Stimulation/no effecta
Stimulation
a
Inhibitiona
a
The effect depends on supply of reducing equivalents and applied mitochondrial substrates.
b
Unspecific side effects of KCO’s not related to activation of mitochondrial
potassium channels.
c
The relative amount of released superoxide depends on activity of SOD1 in the
mitochondrial intermembrane space (cf. [26]).
3
drial superoxide-producing sites, described in the first part of this
review. Additionally, the individual contributions of the different
superoxide-producing sites appear to be tissue dependent [2,8].
The predicted effects of increased potassium influx into the matrix
on ROS production by complexes I and III are summarized in Table 1. This possible site dependency of the observed effect on
superoxide generation has been also discussed by Heinen et al.
[35], who observed upon application of mitoBKCa opener NS
1619 a decrease of reverse electron flow dependent ROS generation
(which is exclusively complex I-dependent), while forward electron flow-dependent ROS generation (at least partially complex
III-dependent) was apparently stimulated by the same potassium
channel opener [33]. More recent observations made with isolated
brain mitochondria [36] indicated however a 20% decrease of
hydrogen peroxide production upon application of the mitoBKCa
openers CGS 7184 and NS 1619, irrespectively of the respiratory
substrates (succinate or glutamate + malate) applied. These effects
were observed to be strictly potassium selective. Moreover, the
specific inhibitors of the BKCa channel, charybdotoxin and iberiotoxin, applied in nanomolar concentrations prevented the observed inhibitory action of the potassium channel openers CGS
7184 and NS 1619 [36]. The differences in the effects observed
by Heinen et al. [33] and by Kulawiak et al. [36] might be related
to different contributions of the complex I- and complex III-dependent superoxide generating sites in heart and brain mitochondria
[8]. Since in brain tissue the complex I-dependent superoxide generation has a higher contribution than the complex III-dependent
superoxide generation [8,9], the observed decrease in superoxide
production is in line with the already mentioned effects of opening
of potassium channels in inner mitochondrial membrane under
resting state conditions of highly reduced NAD-system and of high
resting state 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 leading
to a diminished concentration of FMNH2. This in turn would decrease the production of reactive oxygen species by respiratory
chain complex I (cf. Schemes 1 and 2).
It also needs to be mentioned that studying the mitochondrial
potassium channels is aggravated by the poor selectivity of available potassium channel openers. It appeared that the most widely
used opener of ATP-regulated potassium channel (KATP), diazoxide, apart from stimulating potassium influx into the matrix can inhibit succinate dehydrogenase [37]. NS1619, the popular opener of
calcium-activated potassium channel (BKCa), was shown to inhibit
the activity of respiratory chain complex I [38]. Of course, such direct modulation of the activity of respiratory complexes by KCO’s
strongly affects mitochondrial superoxide generation. In case of
the BKCa channel, the application of iberiotoxin and charybdotoxin, highly selective peptide inhibitors with effective concentrations
in nanomolar range, allows quite reliable inhibitor-based controls.
About the most widely used inhibitor of the mitoKATP channel, 5hydroxydecanoic acid (5-HD) there is much more controversy. In
contrary to the BKCa inhibitors, where the interaction of the drug
with the channel is well described, the mechanism of the mitoKATP inhibition by 5-HD is more elusive. Some observations suggest even, that 5-HD can counteract the consequences of
channel-unrelated effects of diazoxide [37,39]. Thus, while investigating the physiological role of mitochondrial potassium fluxes
with the use of KCO’s a special attention should be paid to the
potassium- and inhibitor-sensitivity of the observed effects and
to the applied opener concentrations. Still, experiments with isolated mitochondria showing that many of the KCO effects can be
reproduced with K+ ionophore valinomycin [32,33] favor the view
that the opener effects are indeed related to activation of mitochondrial potassium channels.
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3. Potential impact of potassium channel mediated alteration of
ROS production on cellular survival
The cytoprotective effect obtained by pre-treatment of tissue
with certain potassium channel openers, like diazoxide, before an
insult is a well known phenomenon [40]. Application of KCO’s before exposing the tissue to ischemia and reperfusion strongly reduces the ischemic injury. A similar effect is obtained by
ischemic preconditioning (IPC), i.e., exposing the tissue to short
ischemic periods before the appearance of longer ischemia and
reperfusion. After the additional discovery, that IPC can be abolished by potassium channel inhibitors, it was suggested that opening of potassium channels is an event appearing in the
preconditioning phase and essential for the observed protective
effect.
The beneficial action of KCO’s was mostly studied in cardiac tissue exposed to ischemia and reperfusion. However, similar cytoprotective effects were observed also in brain and kidney [41,42]
and under different insults, like exposure to hydrogen peroxide
or glutamate [43]. Since potassium channels are present both in
plasma membrane and in inner mitochondrial membrane both
sites can be the targets of KCO action. At present the prevailing
view is that the activation of channels located in mitochondria is
crucial for KCO-mediated cytoprotection [34,40]. Still, the link between modulation of mitochondrial function by KCO’s and cytoprotection is not clear. Different hypotheses explain it as a
consequence of matrix volume modulation, of ‘mild uncoupling’
resulting in limited calcium uptake into the mitochondria and
diminished superoxide generation by the respiratory chain [34]
or, in contrary, of stimulation of mitochondrial ROS production,
which leads to the activation of pro-survival signaling pathways
[40]. The last assumption is supported by observations, that the
KCO-induced preconditioning is abolished by co-administration
of antioxidants and, in consequence, prevention of the opener-induced stimulation of ROS generation [28,44,45]. The KCO-mediated cytoprotection was often accompanied by stimulation of
redox sensitive kinases involved in pro-survival signaling pathways, like ERK [44,46] or PI3K [43]. In these studies the protective
effects of KCO’s were abolished by both kinase inhibitors and ROS
scavengers. Moreover, scavenging free radicals prevented ERK activation, showing that ROS generation is necessary for activation of
this kinase during preconditioning. The relation between decreasing mitochondrial membrane potential, increased ROS generation
and preconditioning was demonstrated by Brennan et al. [47]. In
this study the preconditioning was achieved by application of
low doses of the uncoupler FCCP and the protective effect was
abolished by ROS scavengers.
As discussed in the previous part of this review, the Qo site of
complex III is the most probable superoxide-releasing site at which
superoxide production can be stimulated by increased potassium
influx and subsequent decrease in membrane potential. The
assumption that ROS responsible for the preconditioning are originating from complex III is supported by observations, that the
cytoprotective effects are abolished by application of SOD mimetics, which stimulate dismutation of superoxide to hydrogen peroxide [43,45]. Thus, the signaling molecule activating cytoprotective
pathways seems to be superoxide, which is released by complex III
to the cytosolic compartment (Schemes 1 and 2).
Preconditioning can be also achieved by exposing the tissue to
moderate doses of hydrogen peroxide before the ischemic insult
and interestingly, this H2O2 induced protection was sensitive to
the mitoKATP inhibitor 5-HD [48]. It seems then that in the preconditioning cascade of events, increased ROS levels are not only
the consequence of activation of mitochondrial potassium channels but, in addition, can be responsible for the channel opening.
This view is supported by demonstrations of redox sensitivity of
mitochondrial potassium fluxes [49]. Other studies show the
dependence of mitoKATP activation on protein kinase Ce (PKCe).
Activation and translocation of this kinase to mitochondria is observed during preconditioning [50] and PKCe is in turn known to
phosphorylate the ATP-regulated potassium channel, leading to increased activation of the channel and further stimulation of potassium influx into the matrix [32].
During preconditioning a moderate increase in superoxide generation induced by KCO’s seems to be responsible for the beneficial
effects of the openers. In contrary, the massive ROS production observed during reperfusion and responsible for tissue damage appeared to be limited by KCO pre-treatment [39,40]. Reintroduction of oxygen after the hypoxic period, when respiratory
chain is highly reduced, results in strong stimulation of mitochondrial superoxide generation and this in turn leads to oxidative
damage of the cells. Antioxidant treatment can limit the reperfusion-induced tissue damage, confirming the crucial role of ROS in
this type of cell injury [51]. Thus, diminishing the reperfusion
ROS levels by KCO’s can be an important mechanism responsible
for their cytoprotective properties. In the KCO pre-treated tissue
not only the reperfusion ROS levels were lower, but also oxidative
damage of lipids and both nuclear and mitochondrial DNA were
limited [39,41]. The prevention of oxidative stress exerted by KCO’s
can therefore result from (i) activation of pro-survival signaling
pathways during the preconditioning phase (e.g., activation of
PKD leading to increased expression of SOD [52], as well as (ii)
from diminished complex I-dependent ROS generation due to decreased mitochondrial membrane potential (Scheme 2).
As discussed previously, ‘mild uncoupling’ as potential mechanism to reduce ROS production has been proposed to explain tissue
protection against ROS toxicity [18,19]. Therefore, the data of Facundo et al. [34], Heinen et al. [35] and Kulawiak et al. [36] reporting upon application of BK channel openers a decrease of
mitochondrial ROS production suggest that openers of BK channels
could in principle deploy their cytoprotective action by this particular mechanism.
The assumption that pharmacological preconditioning with
KCO’s is caused by opening of mitochondrial potassium channels
can be questioned due to doubtful specificity of the available
potassium channel modulators. Therefore, it has been suggested
that the diazoxide-mediated cardioprotection can be attributed
rather to inhibition of succinate dehydrogenase (SDH) [53] or the
protonophoric properties of the drug [54] than to activation of
potassium channels. Nevertheless, both mentioned side effects of
KCO’s would modulate mitochondrial ROS production in a similar
way (cf. Scheme 2, dashed arrows). They would lead to inhibition
of complex I-dependent ROS production – important for protection
against oxidative stress in the reperfusion phase – and at saturation of substrate supply potentially to a stimulation of complex
III-dependent ROS production, which could be beneficial for activation of pro-survival signaling pathways in the preconditioning
phase.
4. Concluding remarks
The cytoprotective actions of KCO’s are very likely related to
two different effects on mitochondrial ROS production: (i) Their
potential stimulation of complex III-dependent ROS production
which might be relevant for ROS triggered signal transduction to
the cytosol in the precondition phase and (ii) their established
inhibition of complex I-dependent ROS production in the matrix
compartment occurring in the reperfusion phase. Whether these
effects are directly caused by opening of mitochondrial potassium
channels, like mitoKATP or mitoBKCa, or are linked to possible side
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effects of the applied drugs remains to be elucidated yet. Additionally, the potential impact of KCO-induced changes of ROS levels for
intracellular processes should be much better estimated applying
more precise quantitative measurements of the KCO-induced
changes of mitochondrial ROS release. Paradoxically, during ischemia and reperfusion ROS are responsible for both tissue injury and
preconditioning. Thus, it still remains an important goal to determine which types and which sources of ROS are involved in the
particular, beneficial or deleterious effect. Since the efficacy of general antioxidant treatments, giving promising results in some
experimental systems of ischemia and reperfusion, was so far not
confirmed in clinical practice, there is still a need for the development of cytoprotective treatments targeted to the relevant sources
of ROS.
Acknowledgements
This study was supported by grants of the Deutsche Forschungsgemeinschaft (SFB-TR3 A11 and D12) and the BMBF
(01GZ0308, 01GM0868) to W.S.K.
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