Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume

Article abstract of DOI:10.1074/jbc.M109.017731

Modulation of K⁺ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K⁺ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca2⁺ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K⁺ or H⁺ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK_ATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K⁺ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H₂O₂-induced protein kinase C∈ activation. Rather, increased K⁺ conductance reduced H₂O₂ generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.

Full text of DOI:10.1074/jbc.M109.017731

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 741–750, January 1, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Increased Potassium Conductance of Brain Mitochondria
Induces Resistance to Permeability Transition by Enhancing
Matrix Volume*
Received for publication, May 7, 2009, and in revised form, October 13, 2009 Published, JBC Papers in Press, October 30, 2009, DOI 10.1074/jbc.M109.017731
Magnus J. Hansson‡§1, Saori Morota‡, Maria Teilum‡, Gustav Mattiasson‡, Hiroyuki Uchino¶, and Eskil Elme´r‡ʈ
From the ‡Mitochondrial Pathophysiology Unit, Laboratory for Experimental Brain Research, Department of Clinical Sciences,
§
Lund University, SE-221 84 Lund, Sweden, the Departments of Clinical Physiology and ʈClinical Neurophysiology, Lund University
Hospital, SE-221 84 Lund, Sweden, and the ¶Department of Anesthesiology, Tokyo Medical University, Tokyo 160-0023, Japan
؉
Modulation of K conductance of the inner mitochondrial
membrane has been proposed to mediate preconditioning in
ischemia-reperfusion injury. The mechanism is not entirely
understood, but it has been linked to a decreased activation of
mitochondrial permeability transition (mPT). In the present
phenomenon by which organisms respond with protective
mechanisms to potentially recurring challenges (4). A low dose
of one type of stressful stimulus can also induce resistance to
another, e.g. prior transient hyperthermia can protect against
subsequent forebrain ischemia (5). Understanding and control-
ling these seemingly general endogenous survival responses
might enable a clinical therapeutic opportunity to reduce tissue
damage after cerebral or cardiac ischemia (4, 6, 7). Ischemic
preconditioning mediates both a rapid adaptive response com-
ing into effect within minutes to hours as well as an induced
tolerance occurring over a longer time frame requiring gene
activation and de novo protein synthesis. The former is exten-
sively studied in cardiac ischemia and the latter in cerebral
ischemia (6, 7). Various physiological and chemical triggers can
induce preconditioning, and whereas several mediating path-
ways have been characterized, the final causal effectors remain
more obscure (7).
؉
study K channel activity was mimicked by picomolar concen-
trations of valinomycin. Isolated brain mitochondria were
exposed to continuous infusions of calcium. Monitoring of
extramitochondrial Ca2؉ and mitochondrial respiration pro-
vided a quantitative assay for mPT sensitivity by determining
calcium retention capacity (CRC). Valinomycin and cyclophilin
D inhibition separately and additively increased CRC. Compa-
rable degrees of respiratory uncoupling induced by increased
؉
؉
K
or H conductance had opposite effects on mPT sensitivity.
Protonophores dose-dependently decreased CRC, demonstrat-
ing that so-called mild uncoupling was not beneficial per se. The
putative mitoKATP channel opener diazoxide did not mimic the
effect of valinomycin. An alkaline matrix pH was required for
The inner mitochondrial membrane has been proposed to
contain ATP-sensitive potassium channels (mitoKATP),2 which
are pharmacologically distinct from plasma membrane KATP
and may mediate as well as be end-effectors of the precondi-
tioning effect. Although there is substantial support for
mitoKATP activation in preconditioning, the evidence comes
almost exclusively from studies with pharmacological com-
pounds, mainly diazoxide and 5-hydroxydecanoate (7, 8).
Ischemic preconditioning can be closely mimicked by the KATP
opener diazoxide, which shows a concentration-dependent
selectivity for mitoKATP over plasma membrane KATP (9, 10).
Both ischemic and diazoxide-mediated preconditioning can be
blocked by 5-hydroxydecanoate, which is believed to inhibit
the diazoxide-induced opening of mitoKATP (11). A role of
mitoKATP is also implicated in different models of cerebral
ischemia where pretreatment with diazoxide mediates neu-
ronal protection in a 5-hydroxydecanoate-sensitive manner
(12, 13).
؉
mitochondria to retain calcium, but increased K conductance
did not result in augmented ⌬pH. The beneficial effect of vali-
nomycin on CRC was not mediated by H2O2-induced protein
؉
kinase C⑀ activation. Rather, increased K conductance
reduced H2O2 generation during calcium infusion. Lowering
the osmolarity of the buffer induced an increase in mitochon-
drial volume and improved CRC similar to valinomycin without
inducing uncoupling or otherwise affecting respiration. We
propose that increased potassium conductance in brain mito-
chondria may cause a direct physiological effect on matrix vol-
ume inducing resistance to pathological calcium challenges.
A short non-injurious ischemic insult can greatly reduce the
severity of a subsequent prolonged ischemia, a phenomenon
generally referred to as preconditioning or ischemic tolerance
(1–3). This adaptive response can be seen as a general biological
Activation of the mitochondrial permeability transition
(mPT) phenomenon is considered to participate in cell death
pathways associated with various types of pathological stimuli,
* This work was supported by the Swedish Research Council (reference num-
ber 2008-2634), by the Japanese Ministry of Health and Labor and Welfare
Grant 18591724, by the Swedish Society of Medicine, and by the founda-
tions of Stohne, Segerfalk, and the Royal Physiographic Society in Lund. E.
Elme´r is co-founder and officer of Maas Biolab, LLC and NeuroVive Phar- 2 The abbreviations used are: mitoKATP, mitochondrial ATP-sensitive potas-
maceutical AB, which hold intellectual property rights and develop the use
of cyclosporins as cyclophilin D inhibitors for neurological treatment.
1 To whom correspondence should be addressed: Laboratory for Experimen-
talBrainResearch, BMCA13, LundUniversity, SE-22184Lund, Sweden. Tel.:
46-46-2220605; Fax: 46-46-2220615; E-mail: magnus.hansson@med.lu.se.
sium channels; mPT, mitochondrial permeability transition; CRC, cal-
cium retention capacity; Debio-025 (UNIL025), D-MeAla3EtVal4-cyclo-
sporin; ⌬⌿m, mitochondrial membrane potential; PKC⑀, protein kinase C⑀;
ROS, reactive oxygen species; MPG, N-2-mercaptopropionylglycine; CCCP,
carbonyl cyanide m-chlorophenylhydrazone.
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Mitochondrial K؉ Conductance and Ca2؉ Retention
particularly ischemia-reperfusion injury (14–16). The mPT is Fura 6F (250 nM). The mitochondrial suspensions were infused
also implicated in the pathogenesis of several acute or chronic with 200 nmol of CaCl2/(mg ϫ min)(10 ␮M/min). The start of
neurodegenerative diseases, substantiated by the neuroprotec- calcium uptake was defined as the point where the experi-
tive effects of cyclosporin A, its non-immunosuppressive ana- mental curve deviated from a control curve with 1 ␮M ruthe-
logs, or genetic deletion of the mPT component and cyclo- nium red, blocking mitochondrial calcium uptake through the
sporin A target cyclophilin D in animal models (17–19).
uniporter. CRC was calculated as the amount of infused cal-
Ischemic preconditioning and mitoKATP channel activation cium from the start of mitochondrial calcium uptake until the
have been proposed to afford tissue protection by inhibiting start of maximal calcium release. The redox status of NAD(P)H
mPT activation during reperfusion (8, 10, 20, 21). A signaling was determined qualitatively by following autofluorescence of
pathway linking mitoKATP and mPT has been proposed where NAD(P)H with excitation at 340 nm and emission at 460 nm
ϩ
the increased K conductance after mitoKATP opening alkalin- during calcium infusions. ⌬⌿m was assessed qualitatively by
izes the mitochondrial matrix and increases generation of following fluorescence quenching of rhodamine 123 (100 nM),
H2O2, which in turn activates an mPT-associated PKC⑀ (22, excitation and emission at 490 and 528 nm. Light scattering of
23). An alkaline matrix pH has also been put forward as the mitochondria was detected at 90° and measured at 520 nm.
basis for efficient mitochondrial calcium buffering (24). Fur- Mitochondrial H2O2 generation was detected by following the
ϩ
thermore, prevention of matrix contraction by K -mediated oxidation of 1 ␮M Amplex Red to the fluorescent product res-
volume effects may preserve mitochondrial respiratory capac- orufin in the presence of horseradish peroxidase and superox-
ity and energy transfer during ischemia-reperfusion (25–27).
ide dismutase (0.5 and 20 units/ml, respectively). Excitation
The aims of the present study were to (i) investigate if was set to 560 nm and emission to 590 nm. Known amounts of
ϩ
increased K conductance modulates calcium retention capac- H2O2 were added to establish a calibration curve.
ity (CRC) and activation of mPT in brain mitochondria and (ii)
High Resolution Respirometry—Oxygen consumption of
ϩ
to explore the physiological effects of increased K conduc- mitochondria during calcium infusion was measured under
tance and determine the mechanism by which activation of similar conditions as Fura 6F experiments described above
ϩ
mitochondrial K channels improves mitochondrial resistance except for the continuous decline in oxygen concentration in
to calcium challenges in neuronal preconditioning.
the sealed chamber. Also, 10 ␮M bovine serum albumin was
present in the buffers of the experiments comparing respiration
and CRC in 75 and 125 mM KCl. Studies were performed in an
EXPERIMENTAL PROCEDURES
Isolation of Brain Mitochondria—Adult male Wistar rats Oroboros Oxygraph-2k with a Titration-Injection microPump
(Harlan Scandinavia ApS, Allerød, Denmark) of 325–400 g TIP-2k using DatLab 4 software allowing online respiration rate
were allowed ad libitum access to water and food before use. output with high sensitivity, low noise, and concentration-de-
Animals were normally decapitated after a brief exposure to pendent background correction (Oroboros Instruments, Inns-
halothane to minimize stress, but one animal for each experi- bruck, Austria) (30). CRC in the respiration experiments was
mental setting was decapitated without gaseous anesthesia to calculated as the amount of infused calcium from the start of
ensure that the process did not influence the results. All animal elevated respiration rate until the start of rapid respiration
procedures were approved by the Malmo¨/Lund Ethical Com- decrease.
mittee for Animal Research (M221-03, M44-07). Isolation of
Matrix pH Measurements—Changes in intramitochondrial
cortical non-synaptosomal brain mitochondria was achieved by pH were measured by determining the equilibrium distribution
using a discontinuous Percoll gradient as described previously of the weak acid acetate (31). The uncharged protonated spe-
(28, 29). All steps were carried out under ice-cold conditions. cies (HAc) equilibrates over the mitochondrial membranes.
Ϫ
Protein quantification of mitochondrial suspensions was per- The anion (Ac ) is impermeant and, therefore, accumulates in
formed by the Bradford method using bovine serum albumin as an alkaline compartment, such as the mitochondrial matrix,
standard.
due to continued dissociation and uptake of HAc. The pH dif-
Mitochondrial CRC, NAD(P)H Fluorescence, Membrane ference can be determined by the relationship ⌬pH ϭ log10
Ϫ
Ϫ
Potential (⌬⌿m), Light Scattering, and H2O2 Generation—A Acext/Acm, where ext and m refers to extra-matrix and matrix
luminescence spectrometer LS-50B (PerkinElmer Life Sci- compartments, respectively. A whole preparation of brain
ences) with a temperature controlled cuvette holder (37 °C) was mitochondria (ϳ700 ␮g) was divided in half and incubated in
used for all fluorescence and light-scattering experiments. the ordinary KCl media supplemented with 3H-labeled acetate
Mitochondria (100 ␮g/2 ml) were suspended in 125 mM KCl, 20 (0.5 ␮Ci or 18.5 kBq/ml) and 14C-labeled sucrose (0.05 ␮Ci or
ϩ
mM Tris buffer, pH 7.1, containing 2 mM Pi (K ), 1 mM MgCl2, 1.85 kBq/ml) and 1 mM “cold” acetate and sucrose. [14C]Su-
1 ␮M EGTA, 200 ␮M ATP, and 5 mM NADH-linked respiratory crose does not permeate the inner mitochondrial membrane
substrates malate and glutamate. After the addition of mito- and was used to correct for non-matrix 3H activity. After incu-
chondria, 1 ␮g/ml oligomycin, 50 ␮M ADP, and experimental bation with either 3 pmol/mg valinomycin or vehicle (ethanol)
compounds were added. In experiments evaluating matrix vol- in the presence of adenonucleotides and respiratory substrates
ume effects on CRC, the osmolarity of the buffer was decreased (see above), the suspension was carefully layered on top of 900
by lowering the KCl concentration from 125 to 75 mM. Mito- ␮l of silicone oil AR 110 and centrifuged at 20,800 ϫ g for 2 min,
chondrial calcium uptake and release were monitored by the leaving the mitochondria in a pellet and the aqueous phase
excitation ratio (excitation 340/380 nm, emission 509 nm) of above the silicone oil. Half of the supernatant (500 ␮l) was
the extramitochondrial calcium-sensitive fluorescent probe transferred to new vials. The remaining supernatant and sili-
742 JOURNAL OF BIOLOGICAL CHEMISTRY
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Mitochondrial K؉ Conductance and Ca2؉ Retention
cone oil was carefully but rapidly removed, and the mito-
chondrial pellet was lysed in 500 ␮l of H2O. One series of
experiments was also performed without filtering through sil-
icone oil. Both pellet and supernatant samples were deprotein-
ized with perchloric acid (2% w/v final) (32), and the precipitate
was spun down. Two samples (2 ␮l) of supernatant and 500 ␮l
of the clear pellet solution were transferred to scintillation vials
(HDPE 24 ml, VWR) with 5 ml of Ready Safe scintillation fluid
(Beckman Coulter, Fullerton, CA) and H2O (10:1 final). Sam-
ples were measured in a LS 6500 Scintillation counter (Beck-
man Coulter). Similar experiments were performed with
[3H]H2O for matrix volume estimates but did not yield reliable
values as the signal to noise ratio was far inferior to that of
tritiated acetate. For ⌬pH calculations, a matrix volume of 1
␮l/mg was assumed (33).
Statistical Analyses—All experiments were replicated in at
least four separate mitochondrial preparations. Average results
are presented as the mean Ϯ S.D. and were evaluated with one-
way ANOVA followed by the Bonferroni post hoc test. The
level of statistical significance was set to 5%.
RESULTS
ϩ
Increased K Conductance Induced by Low Concentrations of
Valinomycin Increases the CRC of Brain Mitochondria—The
ϩ
ϩ
lipophilic K carrier valinomycin induces an increased K con-
ductance of the inner mitochondrial membrane and translo-
ϩ
cates K from the intermembrane side to the matrix side elec-
trophoretically as a result of the mitochondrial membrane
potential (⌬⌿m, inside negative) (34). In the present study, vali-
ϩ
nomycin was administered at low concentrations to mimic K
channel activity, and the effect on physiological parameters
of brain mitochondria such as oxygen consumption, ⌬⌿m,
light scattering, NAD(P)H fluorescence, and the ability to
sequester calcium was assayed. Valinomycin at 150 pM (3
pmol/mg of mitochondria) significantly increased the CRC of
brain mitochondria exposed to a continuous infusion of cal-
cium (Figs. 1 and 3). Parallel to the CRC assay, during calcium
infusion, valinomycin prolonged the delay before a rapid de-
crease of NAD(P)H fluorescence and oxygen consumption
(data not shown and Fig. 2, respectively). The dose-response
relationship demonstrated a significant increase of CRC at
150–300 pM, whereas higher concentrations gradually attenu-
ated the positive effect, and 2 nM valinomycin decreased CRC
(Fig. 3).
FIGURE 1. Increased K؉ conductance by valinomycin and inhibition of
cyclophilinDseparatelyandadditivelyincreasebrainmitochondrialcal-
cium retention capacity. Suspensions of isolated brain mitochondria in KCl-
based buffer (100 ␮g/2 ml) were infused with 200 nmol of Ca2ϩ/(mg ϫ min)
(10 ␮M Ca2ϩ/min). Experiments were run in the presence of 200 ␮M ATP, 1
␮g/ml oligomycin, 50 ␮M ADP, and 5 mM malate and glutamate. Additions
were made with 150 pM Kϩ carrier valinomycin (Val) and/or 1 ␮M non-immu-
nosuppressive cyclosporin analog D-MeAla3EtVal4-cyclosporin (Cs, also
called Debio-025 or UNIL025). Vehicle (0.2% ethanol) was present in control
runs (Ctrl). A, CRC of brain mitochondria was determined by following the
fluorescenceratioofFura6F(250nM)reflectingextramitochondrialCa2ϩ con-
ϩ
Increased H Conductance by the Protonophore CCCP or
ϩ
ϩ
Electroneutral K /H Exchange by Nigericin Reduces the Cal-
cium Retention Capacity of Brain Mitochondria—In contrast to
ϩ
the effect of valinomycin, increased H conductance of the
inner mitochondrial membrane by the protonophore CCCP
dose-dependently decreased CRC over all tested concentra- centration. A plateau level is reached when mitochondrial calcium uptake
equals the rate of infused calcium, and rapid calcium release occurs at a cer-
tain threshold considered to reflect activation of mPT. B, CRC was calculated
as the amount of infused calcium from the start of calcium uptake (the time
point where the experimental curve deviated from a control curve with inhib-
tions and completely abolished CRC at 200 nM (Fig. 3). The
lowest tested concentrations of CCCP (25 nM) and valinomycin
(150 pM) both significantly increased basal respiration rate from
ited calcium uptake) to the start of maximal calcium release. Values are the
control (1454.1 Ϯ 202, 1449.5 Ϯ 257, and 647.6 Ϯ 150 pmol
O2/(s ϫ mg), respectively), and there was no significant differ-
ence between the compounds (Fig. 2). Thus, even though the
compounds displayed similar degrees of uncoupling, they
means Ϯ S.D. (␮mol Ca2ϩ/mg mitochondria) of four separate mitochondrial
preparations. The asterisk (*) indicates significant difference compared with
control (vehicle run), and the number symbol (#) indicates significant com-
bined effect of the valinomycin and D-MeAla3EtVal4-cyclosporin compared
with each of the drugs separately.
ϩ
ϩ
exhibited opposite effects on CRC. The electroneutral K /H
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Mitochondrial K؉ Conductance and Ca2؉ Retention
FIGURE2. Highresolutionrespirometryofbrainmitochondriaduringcal-
cium uptake and release. Experiments were performed as described in Fig.
1, except mitochondrial suspensions were analyzed in air-tight chambers.
Oxygen consumption of mitochondria was determined during calcium infu-
sion, and the upper trace displays the O2 concentration (nmol/ml) in the
chamber for a representative control experiment. The lower traces show the FIGURE 3. Dose-related effects of increased K؉ conductance and H؉ con-
real-time O2 consumption rate (pmol O2/s ϫ mg mitochondria) of vehicle ductance or diazoxide on brain mitochondrial calcium retention. Exper-
(Control), 150 pM valinomycin, 1 ␮M D-MeAla3EtVal4-cyclosporin, or 25 nM iments were run as described in Fig. 1, and CRC is expressed as % of control
protonophore CCCP. Basal respiration rates were significantly higher in the (vehicle, 0.2% ethanol or DMSO). The Kϩ carrier valinomycin increased CRC at
presence of valinomycin or CCCP. In all runs calcium infusion induced an low concentrations, but a decrease was seen at the highest tested concentra-
increased rate of respiration from basal levels followed by a rapid decrease in tion. In contrast, all tested concentrations of the protonophore CCCP dose-
respiration, attributed to permeability transition. To confirm altered inner dependently decreased CRC. The K-ATP channel opener diazoxide was with-
membrane permeability, 150 ␮M NADϩ was administered, which increased out effect at 5 ␮M, but higher concentrations decreased CRC. The asterisk
respiration in all groups after calcium infusion. Traces are representative indicates significant difference compared with control.
examples of four separate experiments. Disturbances because of air inlet in
the chamber have been deleted in trace 3 (dashed segment). Quantification of
CRC is provided in Figs. 1 and 3.
rapid calcium release after a prolonged period of calcium
uptake occurs concomitantly with a decrease in light-scattering
exchanger nigericin (5 nM) hyperpolarized the mitochondria
and NAD(P)H fluorescence in brain mitochondria and has,
(data not shown) and significantly decreased the CRC of brain
therefore, been attributed to activation of mPT (24). However,
mitochondria suspended in a buffer with physiological pH (Fig.
the calcium-phosphate complexes formed within the matrix
5A). An increase in ⌬⌿m was, therefore, not sufficient to
during calcium loading interfere with light-scattering and
increase CRC per se.
NAD(P)H fluorescence, and changes in these parameters are
Permeability Transition Limits the Extent of Mitochondrial
not necessarily associated with mPT (35). To obtain a more
Calcium Retention—mPT is classically assayed in light-scatter-
specific correlation between CRC and mPT, the calcium infu-
ing assays measuring matrix volume or configuration changes.
sion experiments were repeated in airtight chambers during
Activation of mPT is usually detected after large bolus addi-
measurements of oxygen consumption (Fig. 2). Mitochondrial
tions of calcium or by calcium loading followed by a second
respiration increased during active calcium uptake and reten-
inducing agent such as an oxidant or protonophore. Although
tion, but at the time, corresponding to rapid calcium release,
the mitochondria behave as though they have certain individual
respiration was severely decreased. The respiratory inhibition
probabilities to undergo mPT, after a large bolus of calcium the
was attributed to loss of NAD(H) from the matrix after mPT.
mPT becomes more of an all-or-nothing phenomenon for the
whole mitochondrial population. A compound that slightly
influences the threshold for mPT activation or interferes with
the calcium loading can, therefore, display a dramatic effect.
Recently, more quantitative assays have been introduced where
either small repetitive calcium doses or, as in the present study,
a continuous infusion of calcium is administered. The strengths
of these latter assays are a lesser degree of sudden bioenergetic
demand for the mitochondria (24) and a quantitative evaluation
of both positive and negative effects of a tested treatment. The
measurement of extramitochondrial calcium provides a func-
ϩ
The addition of exogenous NAD , which is impermeant to the
inner mitochondrial membrane in intact mitochondria,
increased respiration after calcium infusion, indicating that the
mitochondria had undergone mPT (Fig. 2). It was not necessary
to supplement cytochrome c in these experiments to reveal the
ϩ
stimulation of respiration by NAD after mPT, but it may be
necessary in mitochondria isolated from other organs (36). Fur-
ther identifying the mPT as the limiting factor for mitochon-
drial CRC is the finding that the cyclophilin D inhibitor
D-MeAla3EtVal4-cyclosporin (Debio-025) increased CRC in-
tional assay for mitochondrial calcium uptake (Fig. 1A). The dependently and additively to valinomycin (Figs. 1 and 2).
744 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 1•JANUARY 1, 2010

Mitochondrial K؉ Conductance and Ca2؉ Retention
FIGURE 4. How increased K؉ conductance may improve mitochondrial
calcium retention and sensitivity to permeability transition. Schematic
illustrations are shown of some proposed physiological effects caused by
increased Kϩ conductance under conditions where mitochondria buffer an
increased cytoplasmic calcium load. Increased Kϩ conductance by Kϩ chan-
nel activation or by the Kϩ-carrier valinomycin may enhance matrix alkaliza-
tion via increased Hϩ extrusion by the electron transport chain (ETC). The
uptake of phosphate (Pi) and the equilibrium between differently protonated
phosphate groups each depends on pH. An alkaline pH enhances the avail-
ability of PO34Ϫ, enabling formation of calcium phosphate complexes (e.g.
Ca3(PO4)2) from free Ca2ϩ. Enhanced matrix volume due to the osmotic effect
of Kϩ may also lead to increased availability of Pi and enhanced calcium com-
plex formation. The maintenance of low [Ca2ϩ] in the mitochondrial matrix
reduces the sensitivity of mPT and may, therefore, increase CRC. Increased Kϩ
conductance has further been suggested to elevate mitochondrial ROS pro-
duction, which activates PKC⑀ and, through its claimed association with the
mPT pore complex, induces resistance to mPT (22, 44, 53).
Calcium Retention Capacity of Brain Mitochondria Is
Dependent on an Alkaline Matrix pH—A low pH is considered
to stabilize the mPT pore complex in a closed configuration.
This is apparent in mPT assays run under de-energized condi-
tions (37, 38), i.e. under conditions where active calcium uptake
and retention is inhibited. The beneficial effect of acidic pH
in inducing resistance toward mPT was confirmed for de-en-
ergized brain mitochondria in the present study (data not
shown) and has also been demonstrated by others (39). In
contrast, under physiological conditions, an alkaline mitochon-
drial matrix is considered to be the basis for efficient calcium
complexation with phosphate forming inactive calcium phos-
phates and, thus, keeping free [Ca2ϩ] at a regulated low level
(40) (Fig. 4). To further study the role of pH on mitochondrial
calcium accumulation and resistance to mPT, calcium infusion
experiments were run over a wide pH range (Fig. 5A). An acidic
pH (pH 6.6) was detrimental for CRC, as were extreme alkaline
conditions (pH 8.6). Neutral or slightly alkaline conditions were
most favorable for the retention of calcium in brain mitochon-
dria (pH 7.1–7.6). In contrast, mitochondria with a reduced
FIGURE 5. The influence of pH on mitochondrial calcium retention and
measures of ⌬pH. A, experiments were performed as in Fig. 1. The pH of the
buffer was modified between 6.6 to 8.6, and for indicated groups, the pH
gradientover theinnermitochondrial membrane(⌬pH, normally matrix alka-
line) was reduced by the electroneutral Kϩ/Hϩ exchanger nigericin (5 nM). A
neutral or slightly alkaline pH was optimal for CRC in control mitochondria,
whereas pH 7.6–8.1 was most beneficial for mitochondria with reduced ⌬pH.
The asterisk (*) indicates significant difference between groups, and the num-
ber symbol (#) indicates significant difference of nigericin compared with
control. B, shown are calculations of ⌬pH using equilibrium distribution of
tritiated acetate. Calculations were based on quench-corrected and 14C-sub-
tracted 3H dpm in mitochondrial supernatants or pellets from mitochondria
incubated with [3H]acetate and [14C]sucrose with or without 3 pmol/mg vali-
nomycin (Val). ns indicates no significant difference in ⌬pH between groups
(n ϭ 12).
ϩ
ϩ
⌬pH (treated with the electroneutral K /H exchanger nigeri-
cin) displayed the largest CRC at pH 7.6–8.1. Thus, with a matrix pH (Fig. 4). A more alkaline matrix lowers free [Ca2ϩ
nigericin-induced reduction in ⌬pH, a more alkaline pH is through increased uptake and deprotonation of phosphate with
]
required for efficient calcium retention.
a resulting increase in calcium-phosphate complexation. To
ϩ
Increased K Conductance Induced by Valinomycin Does Not test this hypothesis, intramitochondrial pH was measured in a
Cause Increased Alkalization or Reduced Free [Ca2ϩ] in the repeated series of experiments using equilibrium distribution
Mitochondrial Matrix—One possible mechanism whereby in- of tritiated acetate. The radioactivity from tritiated acetate used
ϩ
creased K conductance can enhance CRC is elevation of to calculate ⌬pH was not significantly different in control and
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Mitochondrial K؉ Conductance and Ca2؉ Retention
valinomycin-treated mitochondria. Experiments where mito-
chondria were filtered through silicone oil yielded somewhat
higher values of ⌬pH than experiments without filtration
(Ϫ0.54 Ϯ 0.12 and Ϫ0.39 Ϯ 0.11 for the respective control
runs). A summary of the results with all experiments included is
presented in Fig. 5B.
ϩ
Furthermore, there was no indication that increased K con-
ductance caused reduced matrix [Ca2ϩ]. Extramitochondrial
[Ca2ϩ] was not affected during calcium loading (Fig. 1). In cal-
cium set-point experiments (40), the calcium infusion was
halted, and extramitochondrial [Ca2ϩ] reached a new some-
what lower equilibrium level (data not shown). After the addi-
tion of small amounts of the calcium chelator EGTA in the
presence of 18 mM NaCl, mitochondria gradually released Ca2ϩ
and regained a similar equilibrium level of extramitochondrial
[Ca2ϩ] as before calcium chelation, i.e. the mitochondrial cal-
cium set-point concentration (data not shown). As the activity
ϩ
of mitochondrial Na /Ca2ϩ exchanger is dependent on ⌬pH
and matrix [Ca2ϩ] (41, 42), a difference in these parameters
between control and valinomycin-treated mitochondria would
expectedly influence the calcium set-point concentration as
well as the rate of efflux, but no such differences were observed.
Increased Calcium Retention by Valinomycin Is Not Depen-
dent on Increased ROS and the Associated Activation of PKC⑀—
Garlid and co-workers (23) have described a pathway in iso-
ϩ
lated heart mitochondria by which increased K conductance
through the opening of mitoKATP or by valinomycin triggers an
increase in H2O2. Hydrogen peroxide in turn activates PKC⑀,
which associates with and inhibits mPT. They showed that
openers of mitoKATP or valinomycin inhibited calcium-in-
duced mPT, but the protection could be blocked by the PKC
inhibitor Ro318220 and the ROS scavenger N-2-mercaptopro-
pionylglycine (MPG) (23). In the present study, Ro318330 (0.1
␮M) did not influence the valinomycin-mediated increase of
brain mitochondrial CRC (Fig. 6A). MPG (300 ␮M) decreased
CRC both in control and valinomycin-treated mitochondria,
whereas the antioxidant catalase (2000 units/ml) had no effect,
indicating an unspecific disadvantageous effect of MPG unre-
lated to ROS scavenging. To further study a possible contribu-
tion of ROS generation to the beneficial effect of valinomycin
on CRC, H2O2 generation was monitored during the calcium
infusion experiments (Fig. 6B). The oxidation rate of Amplex
Red to the fluorescent product resorufin was significantly
decreased in the presence of 150 pM valinomycin. Furthermore,
successive additions of 50 pM (1 pmol/mg of mitochondria)
valinomycin to brain mitochondria without infused calcium
lowered basal H2O2 generation in a dose-dependent manner
(data not shown). Hence, increased ROS production and acti-
vation of PKC⑀ did not mediate the increased capacity of brain
mitochondria to accumulate calcium in the present study.
Increasing Matrix Volume Enhances Calcium Retention
FIGURE 6. Increased calcium retention by valinomycin is not blocked by
PKC inhibition or associated with increased H2O2 generation. A, shown is
CRC of brain mitochondria during a continuous calcium infusion as described
in Fig. 1. Mitochondria with 150 pM valinomycin were pretreated with the PKC
inhibitorRo318220(100nM), thefreeradicalscavengerMPG(300 ␮M), or2000
units/ml of the antioxidant catalase. MPG reduced CRC with and without vali-
Capacity without Affecting Respiratory Activity—To investigate
ϩ
effects on matrix volume by increased K conductance and to nomycin, indicating a nonspecific negative effect. The asterisks indicate sig-
nificant difference between groups. B, H2O2-dependent oxidation of Amplex
Red to the fluorescent product resorufin was followed during calcium infu-
sion for control (Ctrl) and valinomycin-treated (Val, 150 pM) brain mitochon-
dria. Inserted numbers display mean H2O2 generation rate in pmol/min/mg.
The asterisk indicates significant difference compared with control. a.u., arbi-
trary units.
separate the volume effects from depolarization and uncou-
pling in relation to CRC, the osmolarity of the suspending
buffer was reduced (by reducing the KCl concentration from
125 to 75 mM). Volume effects were estimated by assaying 90°
light scattering, and the nonspecific ionophore alamethicin was
746 JOURNAL OF BIOLOGICAL CHEMISTRY
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Mitochondrial K؉ Conductance and Ca2؉ Retention
affect respiratory rates before or during calcium uptake (n ϭ 5,
representative traces demonstrated in Fig. 7A).
The increase in respiration induced by valinomycin was
ϩ
attributed to K cycling and not to volume differences (neither
nigericin nor the changes in osmolarity above influenced respi-
ration). The augmented oxygen consumption during mito-
chondrial calcium accumulation was largest at the start of infu-
ϩ
sion with a gradual decline corresponding to a K flux of 900 to
ϩ
350 nmol of K /(min ϫ mg). Similar flux rates were obtained in
the absence of active calcium uptake.
The MitoKATP Channel Opener Diazoxide Does Not Mimic
the Effect of Valinomycin and Reduces Calcium Retention—The
putative mitoKATP channel opener diazoxide was tested at
5–200 ␮M for effects on CRC. Care was taken to follow pre-
viously reported conditions for increasing probability of
mitoKATP detection, i.e. ATP was present in solution before
mitochondria and diazoxide dissolved in DMSO was added
immediately after mitochondria to ensure even distribution
(22). However, one discrepancy was the time frame of the
present experiments compared with typical assays detecting
mitoKATP, which usually monitor the first few minutes after the
addition of mitochondria from isolation medium to experi-
mental buffer (22). Diazoxide did not induce any light-scatter-
ing changes, in contrast to valinomycin, which induced readily
detectable decreases already at 50 pM (data not shown).
Whereas 5 ␮M diazoxide had no effect on mitochondrial cal-
cium handling, 30–200 ␮M dose-dependently decreased CRC
(Fig. 3). The negative effects of diazoxide were probably related
to unspecific effects on mitochondria such as uncoupling of
mitochondria at higher doses (43).
Heart Mitochondria—To evaluate the generability of the
findings in brain mitochondria, key experiments were per-
formed in isolated heart mitochondria. A similar correlation
between matrix volume and CRC was found where an increased
matrix volume increased CRC. Aggravated respiratory uncou-
pling dose-dependently reduced CRC, but the relative impor-
tance of uncoupling versus volume changes on CRC differed
between brain and heart mitochondria (data to be published
elsewhere).
FIGURE7. Theinfluenceofmatrixvolumeonbrainmitochondrialcalcium
retention. Conditions were as described in Figs. 1 and 2 except that 10 ␮M
bovine serum albumin was present. A, respirometry of mitochondria sus-
pended in standard (125 mM, trace 1) or reduced (75 mM, trace 2) concentra-
tions of KCl during a continuous calcium infusion are shown. The KCl concen-
tration was reduced to lower the medium osmolarity and thereby increase
matrix volume. Upper traces are representative examples of changes in O2
concentration, and lower traces depict the corresponding respiration rates
DISCUSSION
ϩ
The main finding of the present study was that increased K
conductance, modeled by low concentrations of valinomycin,
(n ϭ 5). The rapid decrease in respiration rate indicates activation of perme- increases mitochondrial CRC and resistance to mPT in isolated
ability transition. There were no significant differences in basal or calcium-
stimulated respiration rates, but there was significant difference in time to
respiratory inhibition. B, quantification of CRC of brain mitochondria sus-
pended in 125 or 75 mM KCl media. CRC was calculated as the amount of
brain mitochondria. Furthermore, CRC was improved by
reducing the osmolarity of the surrounding buffer, indicating
ϩ
that the beneficial effect of increased K conductance could be
infused calcium from the start of the elevated respiration rate until start of
attributed to a direct physiological effect of an enhanced mito-
rapid respiration decrease. The asterisk indicates significant difference
chondrial matrix volume.
between groups.
ϩ
How Increased K Conductance May Improve Mitochondrial
used to obtain a standardized maximal swelling response (data Calcium Retention and Sensitivity to Permeability Transition—
ϩ
not shown). The reduced KCl concentration and 150 pM vali- Increasing mitochondrial K conductance by valinomycin or
ϩ
nomycin, both, reduced light scattering significantly (12.76 Ϯ by activation of a K channel (such as the putative mitoKATP
)
ϩ
2.2 and 17.1 Ϯ 7.1%, respectively, n ϭ 5–8) with no significant will cause K to enter mitochondria along its electrochemical
difference between the treatments. Calculations of CRC dem- gradient. The drop in membrane potential will accelerate
onstrated a significant increase in CRC in mitochondria sus- NADH oxidation at complex I to rebuild the proton motive
ϩ
ϩ
pended in 75 mM compared with 125 mM KCl (Fig. 7). The force. Increased activity of the K /H exchanger will partially
ϩ
reduced KCl concentration did not, however, significantly counteract the influx of K , and depending on the relative
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 747

Mitochondrial K؉ Conductance and Ca2؉ Retention
ϩ
ϩ
increase of K conductance versus K extrusion, a new equi- ated acetate using a Pi, substrate, and adenine nucleotide-con-
librium will be reached. The previously proposed physiological taining buffer, that no change in ⌬pH or calcium set-point
ϩ
effects of this process, besides a reduction in ⌬⌿m and in- occurs after valinomycin treatment. Thus, increased K con-
creased respiration, are (i) matrix alkalization due to the ductance did not mediate its beneficial effect on CRC through
ϩ
ϩ
K /H exchange, (ii) increased mitochondrial ROS produc- an augmented ⌬pH.
tion, which in turn may activate endogenous protective path-
Mitochondrial ROS Generation and PKC⑀ Activation—Acti-
ways, and (iii) matrix swelling caused by the osmotic effect of vation of PKC⑀ is considered to be a key mediator of ischemic
ϩ
K
(22, 44) (Fig. 4).
preconditioning in the heart (50, 51). Besides a role in activating
Matrix pH and Calcium Retention—Mitochondria take up mitoKATP (52), PKC⑀ has been shown to associate with and
calcium electrophoretically through the uniporter above a cer- inhibit activation of the mPT pore complex (53). Increased
tain external [Ca2ϩ], the so-called calcium set-point (40). The mitochondrial K conductance has been suggested to increase
ϩ
calcium uptake capacity of mitochondria is very large in the mitochondrial ROS production both in vitro and in vivo (44,
presence of physiological levels of ADP and Pi, but the intrami- 54). PKC⑀ is believed to be activated by the elevated ROS,
tochondrial free [Ca2ϩ] is kept low because of formation of desensitizing mPT to activation by calcium (23). The increased
inactive calcium phosphates, e.g. Ca3(PO4)2. The complexation ROS has been proposed to be a direct effect of mitoKATP- or
Ϫ
of calcium requires PO43Ϫ, and both the uptake of H2PO4 and valinomycin-induced alkalization of matrix pH (44). However,
the successive deprotonations to PO34Ϫ vary with matrix pH. in our hands valinomycin consistently decreased detected
The free [Ca2ϩ] in equilibrium with Ca3(PO4)2 would, thus, H2O2 in a dose-dependent fashion, and the K carrier signifi-
ϩ
vary significantly in relation to matrix pH due to Pi availability. cantly lowered the generation of H2O2 during calcium infusion,
A theoretic example for [Ca2ϩ] in equilibrium with Ca3(PO4)2 likely related to the depolarization induced by the compound.
in the presence of 2 mM Pi has been presented by D. G. Nicholls Furthermore, inhibition of PKC⑀ and H2O2 scavenging did not
(40), where [Ca2ϩ] would increase from 2 to 100 ␮M if the inhibit the beneficial effect of valinomycin. Thus, we conclude
matrix pH was reduced from 7.8 to 7.0. Thus, a low matrix pH that this pathway does not seem to be active in our mitochon-
and Pi content will have a dramatic effect on calcium-phos- drial preparations and does not underlie the beneficial effect of
phate formation, and free matrix [Ca2ϩ] may be elevated to an increased K conductance.
ϩ
extent where mPT activation will occur. Our results demon-
Matrix Volume and Calcium Retention—In contrast, in-
strate that an expected reduced ⌬pH, i.e. decreased matrix pH, creasing matrix volume by lowering the KCl concentration of
resulted in a reduced CRC regardless if it occurred concomi- the surrounding medium resulted in an enhanced CRC similar
tantly to an increased ⌬⌿m (as with nigericin) or a decreased to that induced by valinomycin, demonstrating that the benefi-
ϩ
⌬⌿m (as with CCCP). Furthermore, when external pH was cial effect of increased K conductance in brain mitochondria
manipulated in mitochondria with or without nigericin treat- is probably mediated by an enhanced matrix volume. Respira-
ment, an alkaline matrix pH (up to a certain extent) was coupled tion and energy transfer can be modulated by matrix volume
to a high CRC, whereas an acidic to neutral matrix pH resulted (25, 26), but no difference in respiration was observed in mito-
in low CRC. In de-energized mitochondria, where respiration chondria suspended in 75 or 125 mM KCl. Extramitochondrial
and consequently ⌬⌿m-dependent calcium transport is inhib- osmolarity has been shown to influence the sensitivity of liver
ited and/or calcium transport is facilitated by a calcium-iono- mitochondria to mPT, which was attributed to volume-
phore, low pH is a potent inhibitor, and Pi is a main inducer of dependent concentrations of endogenous mPT modulators
mPT (37, 38, 45), and this is the general view of their role in (55). Changes in matrix volume have also been demonstrated to
mPT regulation (46, 47). We demonstrate using high resolution alter the mitochondrial affinity for ADP, where an increased
respirometry during calcium infusion that mPT is the end point matrix volume was associated with reduced ADP affinity (56),
of mitochondrial calcium retention. The determination of CRC but this mechanism would rather sensitize mitochondria to
and the simultaneous monitoring of O2 flux offer reliable and mPT than increase CRC. The expanded matrix volume may,
quantitative assays for mPT in a physiological context where however, hold more Pi and reduce some physicochemical
both negative and positive effects of a given treatment are taken restraints in the protein dense matrix to facilitate calcium com-
into account. The strong pH dependence of brain mitochondria plex formation.
to buffer external calcium loads demonstrate that the effects on
Mild Uncoupling and Calcium Retention—Mild uncoupling
free matrix [Ca2ϩ] by pH and Pi during active mitochondrial of mitochondria, i.e. slightly reducing the proton motive force
ϩ
calcium loading are more important than their effect on the and ⌬⌿m by increasing H conductance by, e.g. protono-
mPT pore components per se.
phores, has been suggested to be a beneficial strategy against
Several previous studies using isolated mitochondria have mitochondrial-mediated cell damage via decreased ROS gener-
demonstrated that valinomycin can increase matrix pH (22, 44, ation and limitation of mitochondrial calcium uptake (57, 58).
48, 49). On the other hand, in a physiological medium, per- Diazoxide has been shown to concentration-dependently (IC50
meant anions such as Pi, which are translocated into mitochon- 65 ␮M) decrease the rate and magnitude of calcium uptake in
dria depending on ⌬pH, may buffer a matrix pH elevation isolated heart mitochondria (58). However, the authors further
ϩ
induced by K flux (48). Pi may also be the main charge-com- reported that diazoxide activated mitochondrial release of cal-
ϩ
pensating anion accompanying mitochondrial K uptake as the cium, a process that was prevented by cyclosporin A. This indi-
mitochondrial matrix volume increases. Indeed, we demon- cates that diazoxide at high concentrations actually sensitized
strate here in equilibrium distribution measurements of triti- the mitochondria toward mPT and the associated calcium
748 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Mitochondrial K؉ Conductance and Ca2؉ Retention
release (which we also demonstrate in the present study) rather preconditioning and ischemic tolerance and could constitute a
novel target for therapeutic intervention.
than afforded protection against mPT. It is likely that these
effects of diazoxide are related to unspecific respiratory inhibi-
tion and depolarization at high concentrations rather than spe-
cific activity of mitoKATP. A hampered mitochondrial function
caused by high dosing of diazoxide or other pharmacological
agents secondarily reducing calcium uptake and retention are
likely not compatible with the high energy demand of neuronal
cells, in particular during pathological situations of increased
metabolic stress and cytoplasmic calcium overload (59).
Acknowledgments—We are grateful to David Nicholls, Andrew
Halestrap, and Carsten Ruscher for methodological input, to Tadeusz
Wieloch for support, and to Fredrik Leeb-Lundberg for use of scintil-
lation counter. Debio-025 (D-MeAla3EtVal4-cyclosporin) was kindly
provided by Debiopharm S.A.
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750 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Metabolism and Bioenergetics:
Increased Potassium Conductance of Brain
Mitochondria Induces Resistance to
Permeability Transition by Enhancing
Matrix Volume
Magnus J. Hansson, Saori Morota, Maria
Teilum, Gustav Mattiasson, Hiroyuki Uchino
and Eskil Elmér
J. Biol. Chem. 2010, 285:741-750.
doi: 10.1074/jbc.M109.017731 originally published online October 30, 2009
Access the most updated version of this article at doi: 10.1074/jbc.M109.017731
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