Cell Biology International 32 (2008) 136e145

www.elsevier.com/locate/cellbi

Effect of magnesium on calcium-induced depolarisation of

mitochondrial transmembrane potential
Peter Racay*
Institute of Biochemistry, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic
Received 4 April 2007; revised 28 June 2007; accepted 27 August 2007

Abstract

An effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential (DJm) was investigated. Depending
on the presence of Mg2þ, addition of Ca2þ to suspension of isolated rat heart mitochondria induced either reversible depolarisation or irrevers-
ible collapse of succinate-driven DJm. Irreversible collapse of DJm, observed in the absence of Mg2þ, was insensitive to Ca2þ chelation, in-
hibition of Ca2þ uptake and increased efflux of Ca2þ from mitochondrial matrix. Based on these data, opening of mPTP in a high-conductance
mode is considered to be a major cause of the Ca2þ-induced irreversible collapse of DJm in the absence of Mg2þ. Involvement of mPTP in the
process of Ca2þ-induced collapse of DJm was further supported by protective effect of both CsA and ADP. Reversible collapse of DJm, ob-
served in the presence of Mg2þ, was sensitive to EGTA, ADP; and inhibition of Ca2þ uptake and increased efflux of Ca2þ from mitochondrial
matrix. This may represent selective induction of a low-conductance permeability pathway. Presented results indicate important role of Mg2þ in
the process of Ca2þ-induced depolarisation of DJm mainly through discrimination between low- and high-conductance modes of mPTP. Minor
effect of Mg2þ on Ca2þ-induced depolarisation of DJm was observed at the level of stimulation of DJm generation and inhibition of mitochon-
drial Ca2þ uptake.
Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Mitochondria; Calcium; Magnesium; Trans-membrane potential

1. Introduction 1966). Mitochondria also play an important role in Ca2þ sig-

The mitochondrion is at the core of cellular energy metab-
olism and is the site of most cellular ATP generation. Mito-
chondrial oxidative metabolism converts the energetic
potential of chemical bonds of different substrates (glucose,
free fatty acids and amino acids) into a proton electrochemi-
cal gradient established across the inner mitochondrial mem-
brane (Mitchel, 1966). The proton electrochemical gradient,
which is proportional to the trans-membrane potential, is in
turn converted to ATP, serves as a ubiquitous and universal
fuel driving the most of physiological processes (Mitchel,
Abbreviations: CsA, Cyclosporin A;; mCICR, Ca2þ-induced release of
2þ
Ca from mitochondria; mPTP, mitochondrial permeability transition pore;
DJm, mitochondrial trans-membrane potential; RuR, ruthenium red.
* Tel.: þ421 43 413 1565; fax: þ421 43 413 6770.
E-mail address: racay@jfmed.uniba.sk
nalling (Pozzan et al., 2000; Gunter et al., 2004) and cell
death (Green and Kroemer, 2004; Leo et al., 2005). Calcium,
the ubiquitous second messenger involved in the regulation of
different physiological processes in all types of mammalian
cells (Berridge et al., 2000), is an important regulator of
mitochondrial function, acting at several levels within the
organelle to stimulate ATP synthesis (Jouaville et al., 1999;
Brookes et al., 2004). Conversely, the deregulation of mito-
chondrial Ca2þ homeostasis is now recognised as playing
a key role in either necrotic or apoptotic cell death under sev-
eral pathological conditions (Crompton, 2000; Duchen, 2000).
A mitochondrial matrix Ca2þ overload can trigger a mitochon-
drial permeability transition pore (mPTP) and cytochrome c
release, leading to cell death (Orrenius et al., 2003). Since
mPTP is permeable to protons and other small ions, induction
of mPTP leads to the depolarisation of the inner mitochon-
drial membrane, consequently affecting ATP production and

1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2007.08.024
 P. Racay / Cell Biology International 32 (2008) 136e145
cell viability (Zoratti and Szabo, 1995; Bernardi et al., 2006).
Mitochondrial Ca2þ overload and consequent mPTP opening
is considered to be involved in the mechanisms of several dis-
eases (Bernardi et al., 2006; Kroemer et al., 2007). Despite
progress regarding independent roles of both Ca2þ and mito-
chondrial dysfunction in diseases, molecular mechanisms by
which Ca2þ can elicit mitochondrial dysfunction remain elu-
sive. Unlike Ca2þ, physiological importance of magnesium is
still matter of debate (Murphy, 2000). Since intracellular con-
centration of free Mg2þ is relatively high (0.5e1 mM), Mg2þ
is unlikely to act as a second messenger by rapid changing its
cytosolic concentration. However, changes in Mg2þ have pro-
found effects on cellular metabolism, structure and bioenerget-
ics. Key enzymes or metabolic pathways, mitochondrial ion
transport, plasma membrane Ca2þ channel activities, ATP-re-
quiring reactions, and structural properties of cells and nucleic
acids are modified by changes in Mg2þ concentration (Romani
and Scarpa, 2000). With respect to mitochondria Mg2þ acts at
several levels too. Stimulation of mitochondrial metabolism
leading to the increase of mitochondrial trans-membrane poten-
tial (DJm) (Panov and Scarpa, 1996; Rodriguez-Zavala and
Moreno-Sanchez, 1998), inhibition of mPTP (Bernardi et al.,
1992) and inhibition of both mitochondrial Ca2þ uptake (Vi-
nogradov and Scarpa, 1973) and Ca2þ efflux (Nakashima
et al., 1982; Wingrove and Gunter, 1986) were documented.
Therefore, the aim of the study was to investigate the effect
of magnesium on calcium-induced depolarisation of DJm.
2. Materials and methods
2.1. Isolation of mitochondria
Mitochondria from rat heart were isolated by differential cen-
trifugation. Adult male Wistar rats were deeply anaesthetised by
inhalation of 2% halothane, 30% O2 and 68% N2O mixture and
sacrificed by rapid decapitation. Dissected tissue was homoge-
nised in ice-cold homogenisation buffer (10 mM TriseHCl
pH ¼ 7.5, 250 mM sucrose, 1 mM EDTA) using Potter
homogenisator. Homogenate was centrifuged at 400  g for
5 m and supernatant was collected. Remaining sediment was
re-homogenised in homogenisation buffer and then centrifuged
at 400  g for 5 m. Both supernatants were pooled and centri-
fuged at 10,000  g for 10 m. The resulting sediment was re-
suspended in isolation buffer (5 mM Hepes-KOH pH ¼ 7.5,
70 mM sucrose, 210 mM manitol, 0.01 mM EGTA, 0.25%
BSA) and centrifuged at 10,000  g for 10 m. The final
sediment was re-suspended in isolation medium and stored on
ice. Protein concentration was determined by Bio-Rad DC
protein assay using BSA as standard.
2.2. Measurement of mitochondrial trans-membrane
potential
The trans-membrane potential of intact heart mitochondria
was measured at 25  C following the safranine O fluorescence
quenching at 540 nm (excitation) and 585 nm (emission) using
137
Shimadzu RF540 spectrofluorophotometer according to Arpa-
gaus et al. (2002). Freshly isolated heart mitochondria (1 mg
of protein) were suspended in 2 ml of air-saturated basal me-
dium (isolation buffer supplemented with 10 mM safranine,
2 mM rotenone, and 1 mg/ml of oligomycin). The effect of
Mg2þ and/or 0.1 mM ADP was investigated by varying their
presence and concentration in incubation medium (see figure
legends). The generation of DJm was induced by the addition
of 5 mM sodium succinate as substrate.
2.3. Mitochondrial Ca2þ uptake
Mitochondrial Ca2þ uptake was measured by rapid filtra-
tion method using 45CaCl2. Isolated mitochondria (0.5 mg of
protein) were suspended in 1 ml of air-saturated incubation
medium (isolation buffer supplemented with 2 mM rotenone,
1 mg/ml of oligomycin, 5 mM succinate). The effect of
5 mM MgCl2 and/or 0.1 mM ADP was investigated by varying
their presence in incubation medium (see figure legends). Af-
ter 3 m of incubation of mitochondria at 25  C in incubation
medium, Ca2þ uptake was initiated by addition of 5 kBq
45
CaCl2/EGTA buffers to yield either 25 mM or 125 mM of
free Ca2þ to incubation medium. Free Ca2þ concentration
was calculated by Cabuf software, kindly provided by Dr. G.
Droogmans (K.U. Leuven, Belgium). Total Ca2þ concentra-
tion was constant being 225 mM. Non-specific Ca2þ transport
was determined using the same conditions and addition of
0.25 mM ruthenium red (RuR) in incubation media. After 2,
4, 6, 8 and 10 m of incubation at 25  C, aliquots of 100 ml
were immediately filtered through Millipore membrane filters
(HA, 0.45 mm), washed with 4  5 ml of ice-cold stop solution
(10 mM TriseHCl pH ¼ 7.4, 180 mM KCl) and dried. Radio-
activity was measured by liquid scintillation spectrometer Tri-
Carb 2200 CA. The number of moles of accumulated calcium
per mg of mitochondrial protein was calculated for each time
point by subtracting values determined in the absence and
presence of RuR. Values of initial rate of Ca2þ uptake and ca-
pacity of mitochondria to accumulate Ca2þ were calculated by
mathematical analysis of measured data assuming first order
kinetics of Ca2þ accumulation.
3. Results
The addition of 25 mM CaCl2 to the suspension of mito-
chondria isolated from rat heart induced collapse of succi-
nate-driven DJm (Fig. 1A). Increased efflux of Ca2þ from
mitochondrial matrix by addition of Ca2þ ionophore
A23187, led to slight repolarisation of DJm, which was
only partially re-polarised by the addition of RuR, mitochon-
drial Ca2þ transport inhibitor. Addition of 5 mM MgCl2 led
to significant but not complete re-polarisation of DJm and
complete re-polarisation was achieved after addition of
0.1 mM ADP (Fig. 1A). ADP alone had no effect on re-polar-
isation of DJm and the presence of Mg2þ seems to be essen-
tial with respect of DJm re-polarisation, (Fig. 1A dashed line).
This is in line with previous findings that ADP alone is unable
to restore collapsed DJm unless Mg2þ is also added
138 P. Racay / Cell Biology International 32 (2008) 136e145

Fig. 1. Effect of A23187 and RuR on Ca2þ-induced depolarisation of DJm. Depolarisation of heart mitochondrial DJm in response to the increase of [Ca2þ] in
basal medium (A), in the presence of 5 mM MgCl2 (B), in the presence of 0.1 mM ADP (C), and in the presence of both 5 mM MgCl2 and 0.1 mM ADP (D). Each
trace represents the changes in DJm monitored as a quenching of Safranine O fluorescence. Efflux of Ca2þ from mitochondrial matrix was initiated by addition of
Ca2þ ionophore A23187 to final concentration of 12.5 mg/ml. Mitochondrial Ca2þ transport was inhibited by addition of ruthenium red (RuR) to final concentration
of 0.25 mM. In order to indicate a complete DJm depolarisation, sodium azide to final concentration of 2 mM was added. The traces are representative of 3 in-
dependent experiments.

(Toninello et al., 1983). In the presence of 5 mM MgCl2, 25
mM CaCl2 induced only partial de-polarisation and pro-
nounced de-polarisation of DJm was achieved by increase
of CaCl2 concentration to 125 mM (Fig. 1B). The addition of
Ca2þ ionophore A23187 led to the significant but not complete
re-polarisation, suggesting that accumulation of Ca2þ within
mitochondrial matrix leading to increased proton permeability
is partially responsible for depolarisation of DJm. Complete
re-polarisation was achieved after inhibition of mitochondrial
Ca2þ uptake by RuR (Fig. 1B), indicating that dissipation of
proton gradient associated with mitochondrial Ca2þ uptake
contributes significantly to depolarisation of DJm. Because
 P. Racay / Cell Biology International 32 (2008) 136e145
addition of ADP stimulates the effect of Mg2þ, the effect of
ADP on Ca2þ induced de-polarisation of DJm was also inves-
tigated. To prevent ADP phosphorylation via mitochondrial
ATPase, oligomycin was included in basal medium. In the
presence of 0.1 mM ADP, 25 mM CaCl2 induced only partial
de-polarisation DJm (Fig. 1C). An increase of CaCl2 concen-
tration to 125 mM led to the collapse of DJm, which was not
affected by addition of A23187 and partial re-polarisation was
achieved after addition of RuR (Fig. 1C). In the presence of
both 5 mM MgCl2 and 0.1 mM of ADP, addition of 125 mM
CaCl2 led to the slight de-polarisation of DJm and significant
de-polarisation but not collapse of DJm was achieved by in-
crease of CaCl2 concentration to 225 mM (Fig. 1D). Increased
efflux of Ca2þ from mitochondrial matrix, initiated by addition
of A23187, had no effect on DJm. The ionophore provided
a pathway for maximal Ca2þ-efflux promoting optimal condi-
tions for maximal Ca2þ cycling and DJm dissipation. As the
mitochondria were fully re-polarised after addition of RuR,
Ca2þ cycling appears to contribute substantially to the sus-
tained Ca2þ-induced de-polarisation in the presence of both
Mg2þ and ADP.
In the following experiments the effect of extra mitochon-
drial Ca2þ chelation on Ca2þ-induced de-polarisation of
DJm was investigated. Collapse of DJm induced by addition
of 25 mM CaCl2 was insensitive to chelation of free Ca2þ
(Fig. 2A) indicating irreversible increase of membrane perme-
ability, which was further insensitive to CsA (data not shown).
Complete re-polarisation was achieved by the addition of
5 mM MgCl2 supplemented with 0.1 mM ADP (Fig. 2A). In
the presence of 5 mM MgCl2, massive de-polarisation of
DJm triggered by addition of 125 mM CaCl2 was almost fully
restored after addition of 1 mM EGTA. Further addition of
0.1 mM ADP led to even more pronounced re-polarisation
(Fig. 2B). In the presence of 0.1 mM ADP, collapse of DJm
induced by addition of 125 mM CaCl2 was insensitive to
Ca2þ chelation. The addition of 1 mM EGTA was not able
to restore DJm, which was completely re-polarised after addi-
tion of 5 mM MgCl2 (Fig. 2C). In the presence of both 5 mM
of MgCl2 and 0.1 mM of ADP, sustained de-polarisation in-
duced by addition of 225 mM CaCl2 was abolished by chela-
tion of extramitochondrial Ca2þ (Fig. 2D).
In the absence of Mg2þ, opening of the high-conductance
mPTP, leading to the translocation of protons and other small
ions back to matrix, represents possible explanation of the
Ca2þ-induced collapse of DJm. As opening of mPTP requires
accumulation of Ca2þ within mitochondrial matrix (Zoratti
and Szabo, 1995; Bernardi, 1999) the effect of both A23187
and RuR pre-treatment was investigated. In addition, effect
of cyclosporine A (CsA), potent inhibitor of mPTP opening
(Broekemeier et al., 1989; Szabo and Zoratti, 1991; Novgoro-
dov et al., 1992) was investigated. Pre-treatment of mitochon-
dria with RuR led to the insensitivity of mitochondria to CaCl2
as it is expected from the fact that RuR as potent inhibitor of
mitochondrial Ca2þ uniporter prevents accumulation of Ca2þ
in mitochondrial matrix (not shown). The effect of both
A23187 and CsA pre-treatment on Ca2þ-induced collapse of
DJm depends on the presence of Mg2þ and ADP. If both
139
Mg2þ and ADP were omitted, pre-incubation of isolated mito-
chondria with A23187 did not protect from collapse of DJm
induced by addition of 25 mM CaCl2. However, DJm was
fully re-polarised after addition of RuR (Fig. 3A), indicating
that dissipation of proton gradient due to mitochondrial
Ca2þ uptake is responsible for DJm collapse. Similarly, che-
lation of extramitochondrial Ca2þ led to the fast and full re-po-
larisation of DJm (not shown). In the presence of ADP, pre-
incubation of mitochondria with A23187 increased resistance
of mitochondria against Ca2þ-induced de-polarisation of
DJm. Addition of 25 mM CaCl2 induced only slight de-polar-
isation of DJm (Fig. 3C). Increase of Ca2þ concentration to
125 mM led to significant and sustained de-polarisation of
DJm. Full re-polarisation was achieved by chelation of ex-
tra-mitochondrial Ca2þ and addition of 5 mM MgCl2
(Fig. 3C). Since pre-treatment of mitochondria with A23187
prevents opening of mPTP, the effect of ADP might be attrib-
utable to both ADP-induced stimulation of mitochondrial res-
piration and ADP-mediated inhibition of mitochondrial Ca2þ
uptake. Pre-treatment of mitochondria with CsA, had a strong
protective effect on mitochondria with respect of Ca2þ-in-
duced collapse of DJm (Fig. 3B). The addition of 25 mM
CaCl2 to the suspension of mitochondria pre-treated with
1.25 mM CsA did not induce collapse of DJm (Fig. 3B). Fur-
ther addition of CaCl2 up to 125 mM led to the significant and
sustained de-polarisation of DJm, which was further en-
hanced after addition of A23187. However, complete re-polar-
isation was achieved after the addition of RuR indicating that
Ca2þ-induced de-polarisation of DJm in the presence of CsA
is due to dissipation of proton gradient associated with mito-
chondrial Ca2þ transport. The addition of ADP to mitochon-
dria pre-treated by CsA did not affect significantly protective
properties of CsA (Fig. 3D). In the presence of either Mg2þ
or Mg2þ in combination with ADP, pre-treatment of mitochon-
dria with CsA did not affect significantly Ca2þ-induced de-po-
larisation of DJm (not shown). This supports the idea that in
the presence of Mg2þ, the opening of high-conductance mPTP
is not involved in the process of Ca2þ-induced DJm de-
polarisation.
In order to estimate the sensitivity of mitochondria to Mg2þ
concentration, the effect of Mg2þ in a concentration dependent
manner was investigated. As Mg2þ alone is not able to give pro-
tection from Ca2þ-induced de-polarisation, all following mea-
surements were done in the presence of 0.1 mM ADP. Three
different concentrations of Mg2þ were chosen, 12.5 mM,
0.25 mM and physiological concentration of 0.8 mM. The pres-
ence of 12.5 mM MgCl2, 25 mM CaCl2 induced only partial de-
polarisation and collapse of potential was achieved by increase
of CaCl2 concentration to 125 mM (Fig. 4A). The addition of
1 mM EGTA led to the partial re-polarisation and complete
re-polarisation was achieved by addition of 5 mM MgCl2. Al-
most the same situation was observed in the presence of
0.25 mM MgCl2, however, chelating of extra-mitochondrial
Ca2þ led to nearly complete re-polarisation of DJm
(Fig. 4B). In the presence of 0.8 mM Mg2þ, DJm de-polarised
after addition of 125 mM CaCl2 and was fully restored after ad-
dition of 1 mM EGTA (Fig. 4C).
140 P. Racay / Cell Biology International 32 (2008) 136e145

Fig. 2. Effect of EGTA on Ca2þ-induced depolarisation of DJm. Depolarisation of heart mitochondrial DJm in response to the increase of [Ca2þ] in basal medium
(A), in the presence of 5 mM MgCl2 (B), in the presence of 0.1 mM ADP (C), and in the presence of both 5 mM MgCl2 and 0.1 mM ADP (D). Each trace rep-
resents the changes in DJm monitored as a quenching of Safranine O fluorescence. Extramitochondrial Ca2þ was chelated by addition of 1 mM EGTA (final
concentration). In order to indicate a complete DJm depolarisation, sodium azide to final concentration of 2 mM was added. The traces are representative of
3 independent experiments.

As the uptake of Ca2þ by respiring mitochondria is driven by
DJm with consequent proton gradient dissipation (Bernardi,
1999), effect of both Mg2þ and ADP on mitochondrial Ca2þ up-
take was investigated. The results are shown in Fig. 5 and sum-
marised in Table 1. Ca2þ uptake by isolated mitochondria
varied substantially on the presence of Mg2þ and ADP. In basal
medium, mitochondria rapidly accumulated Ca2þ, however,
this phase was followed by slow spontaneous Ca2þ release
(Fig. 5A). The release phase timing correlates well with the col-
lapse of DJm, as observed after addition of 25 mM CaCl2 (Figs.
1A and 2A). The addition of 5 mM MgCl2 into basal medium
prevented the release phase and the level of intra-mitochondrial
Ca2þ was almost constant within the interval of 4e10 m
(Fig. 5B). In the presence of 0.1 mM ADP, the rate of Ca2þ
 P. Racay / Cell Biology International 32 (2008) 136e145 141

Fig. 3. Effect of A23187 and cyclosporine A (CsA) pre-treatment on Ca2þ-induced depolarisation of DJm. Depolarisation of heart mitochondrial DJm in response
to the increase of [Ca2þ] to 25 mM and 125 mM Ca2þ in basal medium (A, B), and in the presence of and 0.1 mM ADP (C, D). Isolated mitochondria were first pre-
treated with 12.5 mg/ml A23187 (A, C) or 1.25 mM CsA (B, D) and then the changes in DJm were monitored. Mitochondrial Ca2þ transport was inhibited by
addition of ruthenium red (RuR) to final concentration of 0.25 mM. Extramitochondrial Ca2þ was chelated by addition of 1 mM EGTA. In order to indicate a com-
plete DJm depolarisation, sodium azide in final concentration of 2 mM was added. The traces are representative of 3 independent experiments.

uptake was not significantly affected, however, a significantly 4. Discussion

higher capacity of mitochondria to accumulate Ca2þ was ob-
served (Fig. 5C, Table 1). Similar results were observed after ad-
dition of both Mg2þ and ADP into basal medium (Fig. 5D). An
increase of free Ca2þ to 125 mM led to the increase of both Ca2þ
uptake rate and the capacity of mitochondria to accumulate
Ca2þ (Fig. 5D, Table 1).
The study aimed to investigate the effect of Mg2þ on Ca2þ-
induced de-polarisation of mitochondrial trans-membrane po-
tential. Depending on the presence of Mg2þ, addition of
Ca2þ to the suspension of isolated rat heart mitochondria in-
duced either reversible de-polarisation or irreversible collapse
142 P. Racay / Cell Biology International 32 (2008) 136e145

Fig. 4. Effect of Mg2þ concentration on Ca2þ-induced depolarisation of DJm. Depolarisation of heart mitochondrial DJm in response to the increase of [Ca2þ] to
25 mM and 125 mM Ca2þ in the presence of 12.5 mM MgCl2 (A), 0.25 mM MgCl2 (B), and 0.8 mM MgCl2 (C). Extramitochondrial Ca2þ was chelated by addition
of 1 mM EGTA. In order to indicate a complete DJm depolarisation, sodium azide to final concentration of 2 mM was added. The traces are representative of 3
independent experiments.

of succinate driven DJm. The effect of Mg2þ on reversibility
of Ca2þ-induced de-polarisation has already been documented
(Lotscher et al., 1980) and may indicate two different mecha-
nisms of membrane Ca2þ-induced permeabilisation.
Irreversible collapse of DJm, observed in the absence of
Mg2þ, was insensitive to Ca2þ chelation, inhibition of Ca2þ
uptake and increased efflux of Ca2þ from mitochondrial ma-
trix. Based on these data, opening of mPTP in a high-conduc-
tance mode is possibly a major cause of the Ca2þ-induced
irreversible collapse of DJm in the absence of Mg2þ. It is
well known that opening of mPTP triggered, among other fac-
tors, by accumulation of Ca2þ within mitochondrial matrix
(Zoratti and Szabo, 1995; Bernardi, 1999) is inhibited by
Mg2þ. Involvement of mPTP in the process of Ca2þ-induced
collapse of DJm was further supported by effect of both
CsA and ADP. Pre-treatment of mitochondria with CsA,
a well-known inhibitor of mPTP, protected mitochondria
from irreversible de-polarisation. ADP alone has the potency
to inhibit mPTP via interaction with ADP/ATP translocator,
which is considered to be a part of molecular complex
involved in opening of mPTP (Zoratti and Szabo, 1995;
Bernardi, 1999). The addition of ADP to incubation media in-
creased resistance of mitochondria but did not give protection
from irreversible collapse of DJm. Interestingly, knocking out
of ADP/ATP translocator (Kokoszka et al., 2004) increased re-
sistance of liver mitochondria to approximately 125 mM Ca2þ,
as observed in this study after the addition of ADP to the in-
cubation media. In addition to the inhibitory effect of ADP
on mPTP opening, increased resistance to Ca2þ of A23187
pre-treated mitochondria in the presence of ADP indicate
that effect of ADP is more complex. Since pre-treatment of
mitochondria with A23187 prevents matrix accumulation of
Ca2þ and consequent mPTP opening, dissipation of protons
due to mitochondrial Ca2þ transport is most probably the
main reason of DJm de-polarisation in the case of A23187
pre-treated mitochondria. This is supported by rapid re-polar-
isation of DJm after addition of RuR. It has been documented
that ADP stimulates generation of DJm (Panov and Scarpa,
1996; Rodriguez-Zavala and Moreno-Sanchez, 1998) and in-
hibits mitochondrial uniporter involved in mitochondrial
Ca2þ uptake (Litsky and Pfeiffer, 1997). Increased resistance
to Ca2þ of A23187 pre-treated mitochondria in the presence
of ADP could be therefore attributable to: (1) increased rate
of DJm generation due to ADP-induced stimulation of mito-
chondrial respiratory chain; and (2) decreased dissipation of
protons due to ADP-mediated inhibition of mitochondrial
Ca2þ uptake.
Reversible collapse of DJm, observed in the presence of
Mg2þ, was sensitive to ADP and EGTA; and was dependent
on increased matrix concentration of Ca2þ since inhibition
of Ca2þ uptake or increased efflux of Ca2þ from mitochondrial
matrix led to re-polarisation of DJm. This may represent
selective induction of a Ca2þ-dependent low-conductance per-
meability pathway sensitive to ADP and EGTA as was docu-
mented using succinate-energised rat brain mitochondria
(Brustovetsky and Dubinsky, 2000). Due to the lack of a spe-
cific inhibitor, our knowledge concerning a low-conductance
mode of mPTP were not sufficient. Single-channel recordings
of inner mitochondrial membrane channels with multiple con-
ductance levels suggested that the mPTP has both low- and
high-conductance states (Kinnaly et al., 1989; Szabo and Zor-
atti, 1989). It would appear that operation of the mPTP in the
low-conductance mode plays a role in intracellular calcium
homeostasis and can lead to mitochondrial calcium-induced
calcium release (Ichas et al., 1997). In liver and heart mito-
chondria, the low-conductance state of the mPTP may precede
 P. Racay / Cell Biology International 32 (2008) 136e145 143

Fig. 5. Effect of 5 mM MgCl2 and 0.1 mM ADP on mitochondrial Ca2þ transport. Ca2þ accumulation by heart mitochondria was determined by rapid filtration
method in incubation medium (A), in the presence of 5 mM MgCl2 (B), in the presence of 0.1 mM ADP (C), and in the presence of both 5 mM MgCl2 and 0.1 mM
ADP (D). Total [Ca2þ] was maintained to be 225 mM, concentration of free Ca2þ was set to 25 mM (C) or 125 mM (B) by addition of EGTA. Non-specific Ca2þ
transport was determined using the same conditions and addition of 0.25 mM RuR in incubation media (,). Results are shown as mean  standard deviation of
three independent measurements.

and remain open after closure of a Ca2þ-induced large conduc-
tance pore (Al-Nasser and Crompton, 1986; Broekemeier
et al., 1998). However, it is not yet clear whether the same
complex of proteins is responsible for high- and low-conduc-
tance mPTP.
In the presence of Mg2þ and ADP, Ca2þ-induced de-polar-
isation was not likely to be caused by opening of mPTP in the
low-conductance mode. This concurs with finding that low-
conductance permeability is inhibited by ADP (Brustovetsky
and Dubinsky, 2000). Presented data indicate that Ca2þ-in-
duced de-polarisation in the presence of both Mg2þ and
ADP was most probably due to Ca2þ cycling. Operation of
Ca2þ cycling was further supported by the fact that addition
of A23187 had no effect on repolarisation of DJm, which
was fully restored after inhibition of Ca2þ uptake by RuR.
Ca2þ cycling occurs after matrix Ca2þ concentration increases
144 P. Racay / Cell Biology International 32 (2008) 136e145
Table 1
Effect of 5 mM MgCl2 and 0.1 mM ADP on parameters of mitochondrial Ca2þ
transport
Initial rate Capacity to
of Ca2þ accumulation accumulate Ca2þ
(nmol of Ca /mg min) (nmol of Ca2þ/mg)
2þ
Basal medium y27.2 Not determined
(25 mM free Ca2þ)
5 mM MgCl2 66.2  4.9 77.3  0.9
(25 mM free Ca2þ)
0.1 mM ADP 72.2  3.8 111.9  1.1
(25 mM free Ca2þ)
0.1 mM ADP þ 5 mM MgCl2 80.7  5.2 97.8  1.0
(25 mM free Ca2þ)
0.1 mM ADP þ 5 mM MgCl2 192.2  13.6 196.3  1.9
(125 mM free Ca2þ)
Ca2þ uptake by isolated heart mitochondria was determined by rapid filtration
method in incubation medium, in the presence of 5 mM MgCl2, in the pres-
ence of 0.1 mM ADP, and in the presence of both 5 mM MgCl2 and
0.1 mM ADP. Total Ca2þ concentration was 225 mM; concentration of free
Ca2þ was set to 25 mM or 125 mM by addition of EGTA. The initial rate of
Ca2þ accumulation and capacity of mitochondria to accumulate Ca2þ was cal-
culated by numerical analysis of results presented on Fig. 5 assuming first or-
der of Ca2þ accumulation.
above a certain level and plays an important role in modula-
tion of intracellular Ca2þ transients (Bernardi, 1999). In this
study, Ca2þ cycling was observed in two different conditions
(in Mg2þ free media after pre-incubation of mitochondria
with A23187 and in the presence of both Mg2þ and ADP),
having different impacts on DJm. In the case of A23187
pre-treated mitochondria already 25 mM Ca2þ led to almost
complete de-polarisation of DJm, addition of 225 mM Ca2þ
caused only partial depolarisation of DJm in the presence of
both ADP and Mg2þ. Such a difference might be attributable
to the effect of Mg2þ and ADP on both mitochondrial Ca2þ
transport and generation of DJm. It is well known that the
rate of mitochondrial Ca2þ uptake is proportional to DJm
and energy in the form of proton electrochemical gradient is
required for mitochondrial Ca2þ uptake (Bernardi, 1999).
The rate of Ca2þ accumulation in the absence of both Mg2þ
and ADP was the slowest among different conditions tested.
However, accumulation of Ca2þ is significantly decreased
due to leak of Ca2þ from mitochondria through mPTP. Thus
the rate of Ca2þ transport from cytoplasm to matrix might
be high enough to de-polarise DJm completely. On the other
hand, the ability of Mg2þ to inhibit Ca2þ currents mediated by
putative Ca2þ uniporter has been documented recently (Kiri-
chok et al., 2004). It has also been documented that Mg2þ
stimulates generation of DJm (Panov and Scarpa, 1996;
Rodriguez-Zavala and Moreno-Sanchez, 1998). It is likely
that both increased DJm generation together with decreased
rate of Ca2þ uptake and thus decreased proton dissipation
are most probably reflected by partial de-polarisation of
DJm observed in the presence of both Mg2þ and ADP.
Since mitochondria are excitable organelles capable of
generating and conveying electrical and Ca2þ signals (Ichas
et al., 1997), important question arises about physiological
implication of magnesium influence on Ca2þ-induced DJm
de-polarisation. Ca2þ-induced release of Ca2þ from mitochon-
dria (mCICR) depends on transitory opening of the mPTP op-
erating in the low-conductance mode (Ichas et al., 1997). The
Ca2þ fluxes taking place during mCICR are a direct conse-
quence of the mitochondrial de-polarisation spike caused by
mPTP opening. Results presented in this study raised the pos-
sibility that Mg2þ is keeping mitochondria in the conditions
allowing reversible opening of low-conductance state and
thus to follow cytoplasmic Ca2þ transients. In line with previ-
ous results (Tassani et al., 1995), the low level of Mg2þ, which
is able to protect DJm collapse, indicates high affinity of pu-
tative Mg2þ target. Despite documented fluctuation of Mg2þ
due to different conditions (Romani and Scarpa, 2000), it is
unlikely that the effect of Mg2þ on Ca2þ-induced de-polarisa-
tion is playing an important role in cell signalling. The ques-
tion of the physiological importance of mPTP opening is
complicated by the fact that most measurements of Ca2þ-in-
duced de-polarisation and/or mPTP opening are done in vitro
using suspension of isolated mitochondria, conditions far from
real physiology. There are only a few reports indicating mPTP
opening in vivo and indirect observations about involvement
of mPTP in synaptic plasticity and long-term potentiation
were published recently (Weeber et al., 2002; Levy et al.,
2003). Unlike normal physiology, it is generally believed
that opening of mPTP is implicated in mechanisms of major
human diseases (Bernardi et al., 2006; Kroemer et al.,
2007). For example, ischemia of both heart (Halestrap et al.,
2004) and brain (Starkov et al., 2004) leads to the Ca2þ over-
load and consequent opening of mPTP. This transition causes
mitochondria to become uncoupled and capable of hydrolys-
ing rather than synthesising ATP. Unrestrained, this will lead
to the loss of ionic homeostasis and ultimately necrotic cell
death. In addition to its role in necrosis, transient opening
of the mPTP may occur and lead to the release of cytochrome
c and other proapoptotic molecules that initiate an apoptotic
cascade. Only if subsequent mPTP closure occurs will ATP
levels be maintained, ensuring that cell death continues
down the apoptotic, rather than the necrotic pathway. Results
presented here and published by Eskes and co-workers might
indicate the possible role of Mg2þ in the regulation of cell
death pathways. While Mg2þ exhibits protective effect in
the process of Ca2þ-induced mPTP, Bax-induced cytochrome
c release is facilitated by Mg2þ (Eskes et al., 1998). Thus by
closing mPTP and maintaining ATP levels in association with
Bax-induced cytochrome c release, Mg2þ might play a role
in discriminating between two different cell death pathways
in favour for apoptosis. Interestingly, involvement of cyclo-
philin-D, putative mPTP component, in the regulation of
the cell death pathway mechanism has been documented re-
cently using both knockout mice (Nakagawa et al., 2005;
Baines et al., 2005) and stable transfection approach (Li
et al., 2004).
In conclusion, the results illustrate the important role of
Mg2þ in the process of Ca2þ-induced depolarisation of DJm
mainly through discrimination between low and high conduc-
tance modes of mPTP. A minor effect of Mg2þ on Ca2þ-in-
duced de-polarisation of DJm was observed at the level of
 P. Racay / Cell Biology International 32 (2008) 136e145
stimulation of DJm generation and inhibition of mitochon-
drial Ca2þ uptake.
Acknowledgements
This work was supported by the Ministry of Education of
Slovak Republic (grant VEGA 1/1192/04).
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