Báo cáo khoa học: Mitochondrial Ca2+ sequestration and precipitation revisited docx

MINIREVIEW
Mitochondrial Ca
2+
sequestration and precipitation
revisited
Christos Chinopoulos and Vera Adam-Vizi
Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Budapest,
Hungary
Why is it important to address Ca
2+
sequestration and precipitation?
Isolated mitochondria from a variety of sources exhibit
a finite capacity to accumulate and retain divalent
cations, including Ca
2+
[1,2]. This capacity differs
among mitochondria isolated from various tissues, and
even among regions of the same tissue, for example in
brain [3]. Furthermore, the mitochondrial Ca
2+
accu-
mulation capacity in a tissue may change with age
without measurable bioenergetic alterations [4–6]. The
ability of mitochondria to act as ‘firewalls’ of intracel-
lular Ca
2+
waves enables cells to survive an elevated
[Ca
2+
] emergency [7], and the factors that determine
the capacity of mitochondrial Ca

2+
buffering thereby
affect the cell’s fate. In addition, Ca
2+
uptake capacity
may significantly decrease in pathological settings [8].
The factors that determine this capacity can be classi-
fied as (a) those that define the quantity of Ca
2+
that
can be retained in the mitochondrial matrix, and (b)
those that define the threshold for induction of Ca
2+
release. There is no precedent for a lack of interaction
between factors of the two classes. For both classes,
these factors can be further categorized as ‘intrinsic’
and ‘extrinsic’. ‘Extrinsic’ factors include those that
can be manipulated by experimental conditions, such
as the presence and amount of phosphate and adenine
nucleotides, pH and ionic strength of the medium.
‘Intrinsic’ factors refer to those that result in the vari-
ous Ca
2+
accumulation capacities among the various
types of mitochondria studied under identical experi-
mental conditions. The intrinsic factors also include
Keywords
adenine nucleotides; Ca
2+
uniporter;

complexation; electron microscopy;
Na
+
/Ca
2+
exchanger; phosphocitrate;
polyphosphate; precipitation;
thermodynamics; uncoupler
Correspondence
C. Chinopoulos, V. Adam-Vizi, Department
of Medical Biochemistry, Semmelweis
University, Budapest, Hungary
Fax: +361 2670031
Tel: +361 4591500 ext. 60024;
+361 2662773
E-mail: ;

(Received 9 February 2010, revised 19 May
2010, accepted 22 June 2010)
doi:10.1111/j.1742-4658.2010.07755.x
The ability of mitochondria to sequester and retain divalent cations in the
form of precipitates consisting of organic and inorganic moieties has been
known for decades. Of these cations, Ca
2+
has emerged as a major player
in both signal transduction and cell death mechanisms, and, as a conse-
quence, the importance of mitochondria in these processes was soon recog-
nized. Early studies showed considerable effort in identifying the
mechanisms of Ca
2+

sequestration, precipitation and release by uncouplers
of oxidative phosphorylation; however, relatively little information was
obtained, and these processes were eventually taken for granted. Here, we
re-examine: (a) the thermodynamic aspects of mitochondrial Ca
2+
uptake
and release, (b) the insufficiently explained effect of uncouplers in inducing
mitochondrial Ca
2+
release, (c) the thermodynamic effects of exogenously
added adenine nucleotides on mitochondrial Ca
2+
uptake capacity and
precipitate formation, and (d) the elusive nature of the Ca
2+
-phosphate
precipitates formed in the mitochondrial matrix.
Abbreviations
ANT, adenine nucleotide translocase; PTP, permeability transition pore.
FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS 3637
those that are responsible for emerging differences in
Ca
2+
accumulation capacities upon alterations in the
extrinsic factors. For example, under otherwise identi-
cal experimental conditions, inclusion of adenine nucle-
otides increases Ca
2+
accumulation capacity in rat
brain mitochondria by a factor of $ 20, while that in

rat liver mitochondria is increased by a factor of < 2.
Given the intimate relationship between mitochondrial
Ca
2+
handling and signal transduction and cell death
pathways [9–13], it is imperative to identify the mecha-
nism(s) that define mitochondrial Ca
2+
accumulation
capacity. This will lead to a better understanding of
the contribution of mitochondria to Ca
2+
homeostasis
in physiological and pathological settings, and provide
the opportunity to identify potential targets for phar-
macological ⁄ genetic manipulation. The purpose of this
review is to address the factors that define mitochon-
drial Ca
2+
retention: those affecting the induction of
Ca
2+
release with respect to opening of the permeabil-
ity transition pore (PTP) have been extensively
reviewed elsewhere [9,14] and in the accompanying
reviews [15,16].
Thermodynamic aspects of
mitochondrial Ca
2+
uptake and

release mechanisms
As mentioned above, mitochondria contribute to signal
transduction by sequestering Ca
2+
and releasing it to
the cytosol in a controlled fashion. This is achieved by
Ca
2+
influx and efflux pathways, namely the uniporter,
PTP, the Na
+
⁄ Ca
2+
exchanger, diacylglycerol-sensitive
cationic channel(s), and other less-well characterized
entities [17–19]. As soon as the extra-mitochondrial
[Ca
2+
] exceeds a so-called set-point value [20], Ca
2+
uptake by the uniporter, driven by the large electrical
potential difference (DW
m
= )150 to )220 mV),
results in accumulation of Ca
2+
in the matrix. The
molecular identity of the mitochondrial Ca
2+
uniporter

is still unknown. A recent study suggested uncoupling
proteins 2 and 3 as possible components of the uni-
porter [21], but this has been challenged [22] (for the
response of Graier’s laboratory, see [23]). Even though
the uniporter has been patch-clamped [24], current
state-of-the-art methodologies stop short in the mole-
cular identification of a single protein that can be
captured at the tip of a glass electrode.
The mitochondrial matrix can accumulate Ca
2+
up
to 1 m (free and bound); however, the free [Ca
2+
]in
this compartment ([Ca
2+
]
m
) is within the low micro-
molar range [25]. This results in maintenance of only a
minor chemical gradient for Ca
2+
across the inner
mitochondrial membrane [26]. Substantially higher
transient [Ca
2+
]
m
elevations have been reported using
matrix-targeted aequorins [27]; however, the calibra-

tion techniques used were subsequently criticized [28].
Mitochondrial Ca
2+
uptake has recently been
described as relying exclusively on electrochemical diffu-
sion [29]; however, this is inaccurate. According to the
model by Gunter and Sheu [29], mitochondrial Ca
2+
uptake should be essentially null if DW
m
is more positive
than )120 mV, a prediction that has repeatedly been
experimentally rejected. The accumulation of Ca
2+
by
mitochondria takes precedence over oxidative phos-
phorylation [30], and persists even when mitochondria
are severely depolarized [31] (see below). A kinetic
model of the uniporter that fits the majority of experi-
mental results on mitochondrial Ca
2+
uptake has been
developed recently [32]. This model assumes a six-state
catalytic binding and Eyring’s free-energy barrier the-
ory-based transformation mechanisms, associated with
carrier-mediated facilitated transport and electro-diffu-
sion. The results of this modeling and how the model fits
experimental data on Ca
2+
uptake in isolated respiring

mitochondria from rat liver are shown in Fig. 1A.
In sufficiently energized mitochondria, the release of
sequestered Ca
2+
is thermodynamically unfavorable,
but it is possible via the Na
+
⁄ Ca
2+
exchanger, or
due to the concerted action of the H
+
⁄ Ca
2+
and H
+
⁄
Na
+
exchangers [33]. The activity of the Na
+
⁄ Ca
2+
exchanger in mitochondria was documented 36 years
ago [34], but its molecular identity remained unknown
until recently [35]. A mitochondrial H
+
⁄ Ca
2+
exchan-

ger termed Letm1 has also been identified recently, but
its role in Ca
2+
extrusion is not clear [36]. The identity
of the H
+
⁄ Na
+
exchanger is still unknown.
The thermodynamic equilibrium of the mitochondrial
Na
+
⁄ Ca
2+
exchanger for cytosolic [Na
+
] as a function
of DW
m
has been derived in [37] and is shown in Fig. 1B
(black line). At a given DW
m
, the mitochondrial
Na
+
⁄ Ca
2+
exchanger operates in forward mode, i.e. it
brings Ca
2+

into the matrix in exchange for Na
+
, i.e.
when the concentration of extra-mitochondrial Na
+
falls below the black line within the dark-grey area. The
mitochondrial Na
+
⁄ Ca
2+
exchanger operates in reverse
mode at a given DW
m
, i.e. brings Ca
2+
out of the
matrix in exchange for Na
+
, when the concentration of
extra-mitochondrial Na
+
falls above the black line
within the light-grey area. Based on this model, it is
immediately apparent that > 36 mm Na
+
need to be
added to well-polarized mitochondria (i.e.
DW
m
= )170 mV) in order to induce release of matrix

Ca
2+
by reversal of the mitochondrial Na
+
⁄ Ca
2+
exchanger. Without such an increase in cytosolic [Na
+
],
transient losses of DW
m
are essential for the release of
sequestered Ca
2+
. Such transient losses of DW
m
have
Mitochondrial Ca
2+
precipitation C. Chinopoulos and V. Adam-Vizi
3638 FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS
been described in cells, and are attributed to low-con-
ductance pore openings [38–40].
The effect of uncouplers on Ca
2+
release from mitochondria
As discussed below, sequestered Ca
2+
complexes with
phosphate as well as other molecules including pro-

teins and ribose [41], forming an insoluble precipi-
tate that is in equilibrium with a soluble pool of
Ca
2+
-phosphate complex [25]. In the absence of Na
+
,
dissipation of DW
m
is a prerequisite for Ca
2+
release;
however, this is not sufficient in itself [31]. Addition of
an uncoupler of oxidative phosphorylation, or any
compound that forms a pore in the inner mitochon-
drial membrane, is also required to release sequestered
Ca
2+
. The ability of a pore-opening substance to
affect Ca
2+
release is evident; however, the effect of
an uncoupler requires further clarification. The ability
of the uncoupler 2,4-dinitrophenol to cause simulta-
neous discharge of Ca
2+
and phosphate in the medium
from Ca
2+
-loaded mitochondria was reported 46 years

ago [42], but the first attempt to explain this phenome-
non was made in 2003 [25]. These authors offered
the explanation that acidification results in dissolution
of the Ca
2+
-phosphate complex, allowing free Ca
2+
to leave the matrix, provided that DW
m
is suffi-
ciently diminished. A crucial aspect of the sequence of
events is that the concentration of the PO
3À
4
species,
which is required for complexation of Ca
2+
,is
dependent on the third power of DpH at constant
external phosphate [26].
However, the DpH across the inner mitochondrial
membrane is inversely related to the amount of P
i
in
the medium [43–47], and DpH is in the range 0.11–0.15
in the presence of abundant P
i
[31] (see Fig. 2A). DpH
also remains relatively constant within a range of pH
o

[47] (Fig. 2B). If matrix acidification does indeed
underlie dissolution of the Ca
2+
-phosphate complex
and Ca
2+
release by uncouplers, then complete depo-
larization by combined inhibition of the respiratory
chain plus reversal of the F
0
F
1
-ATPase would have a
similar effect in acidic pH. By the same token, com-
plete depolarization by uncoupling in alkaline pH
should impede dissolution of this complex, thus
impairing the release of sequestered Ca
2+
. The experi-
mental findings [31] do not support the above expecta-
tions, implying that matrix acidification by uncouplers
cannot be the sole explanation for the release of
sequestered Ca
2+
. Nonetheless, the rationale of Chal-
mers and Nicholls is viable, but to what extent? The
calculations used to estimate the intra-mitochondrial
free [Ca
2+
] increase upon pH change caused by pro-

tonophores rely on a hypothetical DpH of 1, prior to
addition of an uncoupler [31]. However, at DpH
<0.15, the uncoupler still releases all the sequestered
Ca
2+
from mitochondria. Using the standard analyti-
cal calculations described previously [31], the amount
of Ca
2+
that can be released is predicted, but not pro-
ven, to be a very minor fraction of the total. In order
to measure experimentally the amount of Ca
2+
that
can be released due to a collapse of DpH, the effect of
nigericin, a H
+
⁄ K
+
antiporter eliminating the DpH in
high-K
+
ionic strength medium must be compared to
that of an uncoupler in Ca
2+
-loaded mitochondria
with no DW
m
. Such an experiment is shown in Fig. 2C.
In curve ’a’, addition of nigericin to mitochondria

completely depolarized by stigmatellin and oligomycin,
A
B
Mito NCX forward
3Na
+
3Na
+
Ca
2+
Ca
2+
Matrix
Matrix
Mito NCX reverse
Fig. 1. (A) Fitting of the Ca
2+
uniporter model (lines) [32] to experi-
mental data on Ca
2+
uptake in isolated respiring mitochondria from
rat liver. Reprinted from [32] with permission from Elsevier. (B)
Thermodynamic equilibrium of the mitochondrial Na
+
⁄ Ca
2+
exchan-
ger. The curve was calculated using an exchange ratio of 3, and
the following concentrations were assumed: Ca
2þ

c
=54nM [37],
Ca
2þ
m
=1lM [25] and Na
þ
m
= 11.4 mM [37] using the resting cyto-
solic concentration from [37]. This figure was used with permission
from Dr Akos A. Gerencser, Buck Institute, Novato, CA, USA.
C. Chinopoulos and V. Adam-Vizi Mitochondrial Ca
2+
precipitation
FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS 3639
caused the release of $ 20% of the total Ca
2+
previ-
ously taken up. Under these conditions (both DW
m
and DpH are essentially zero), subsequent addition of
uncoupler still causes complete loss of sequestered
Ca
2+
from mitochondria (see also curve ‘b’). There is
no plausible explanation for this phenomenon based
on current understanding of the chemiosmotic theory.
Interpretation of the results described above may ben-
efit from the observations by Kristian et al. [48] showing
that a large fraction of sequestered Ca

2+
is retained
even after complete depolarization by uncouplers or
induction of the PTP in isolated [48–52] and in situ
[53,54] mitochondria. Other studies have supported the
view that sequestered Ca
2+
is released from various
matrical Ca
2+
pools, implying matrical micro-compart-
mentation that could promote selective Ca
2+
release
[55–58]. An interaction of Ca
2+
with pyridine nucleo-
tides in non-polar environments has also been proposed
[59–63]. However, these reports appeared before the rec-
ognition of PTP and its regulation by the mitochondrial
redox state [64,65], and the release of sequestered Ca
2+
by oxidation of the NADH pool could be attributed pri-
marily to opening of the PTP. Finally, it is worth men-
tioning that Ca
2+
exhibits the ability to form a complex
with carboxylic acids, many of which are abundant in
the mitochondrial matrix [66]. The concentrations of
carboxylic acids (several of which are substrates ⁄ prod-

ucts of the citric acid cycle) fluctuate, and they exhibit
unequal affinities for Ca
2+
, so it is difficult to predict
the matrix [Ca
2+
]
free
at any given time.
The finding that stigmatellin plus oligomycin
induced robust Ca
2+
release at pH
o
= 7.8, in the
absence of measurable changes in light scattering,
deserves further attention [31]. As there were no
changes in the light scatter recordings, we were poised
to accept that Ca
2+
is released through the uniporter.
To this end, it is to be noted that the uniporter is sub-
ject to activation ⁄ inactivation [67–70], a phenomenon
that is poorly characterized. Experimental conditions
that promote matrix alkalinization significantly reduce
uniporter inactivation, whereas acidification allows
A
B
C
Fig. 2. (A) The influence of DpH (given as Dlog [acetate]) and DW

m
(given as Dlog[Rb
+
]) on the internal and external ATP ⁄ ADP ratio as
a function of added P
i
. The DpH decreases between 0.8 and 0.1 on
increasing P
i
to 10 mM, but DW
m
remains largely constant. Rep-
rinted from [43] with permission from Wiley-Blackwell. (B) Correla-
tion of matrix pH to the pH of the experimental medium, before
and after collapse of DW
m
by the uncoupler SF 6847. Reprinted
from [47] with permission from Elsevier. (C) Reconstructed time
courses of extra-mitochondrial [Ca
2+
], calculated from calcium
green 5N fluorescence. Mitochondria were added at 50 s, followed
by addition of 10 l
M oligomycin at 285 s and 20 lM CaCl
2
at 300 s,
and additions of 1.25 n
M stigmatellin (stigm) as indicated by the
arrows, for both traces. After the 8th addition of stigmatellin, mito-
chondria were completely depolarized (not shown). Nigericin (5 l

M,
trace ‘a’) or SF 6847 (100 n
M, trace ‘b’) were added at 750 s, and
150 n
M SF 6847 was subsequently added to both traces at 1100 s.
Mitochondrial Ca
2+
precipitation C. Chinopoulos and V. Adam-Vizi
3640 FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS
the uniporter to conduct Ca
2+
less readily [71]. As a
Ca
2+
-selective channel, it is not surprising that the
uniporter is gated by protons, something which is
widely recognized for many Ca
2+
-selective channels
[72–74]. It is also known that the F
1
F
0
-ATPase com-
plex is an important source of protons for inactivation
of the uniporter [71]. Proton coupling between the
F
1
F
0

-ATPase and the uniporter channel could account
for the fact that matrix Ca
2+
is released the presence
of stigmatellin and oligomycin at pH
o
= 7.8. The
collapse of DW
m
, combined with inhibition of the
F
1
F
0
-ATP synthase complex by oligomycin in the pres-
ence of a strongly alkaline matrix, could underlie the
de-inhibition of the uniporter, allowing matrix Ca
2+
to be released.
Thermodynamic aspects of the effect
of exogenously added adenine
nucleotides on mitochondrial Ca
2+
uptake capacity
As discussed above, the presence of adenine nucleotides
increases the Ca
2+
accumulation capacity of mitochon-
dria. This has been explained previously as the result of
two effects: (a) adenine nucleotides decrease the thresh-

old for induction of PTP and therefore allow greater
amounts of Ca
2+
to be sequestered [75], and (b)
adenine nucleotides participate in formation of the
matrix Ca
2+
-phosphate precipitates, thus increasing
the amount of Ca
2+
that can be retained [41,48,76].
The effect of adenine nucleotides is thought to be
mediated by binding to either an atractylate-sensitive
site, i.e. the adenine nucleotide translocase, or an
atractylate-insensitive site, the identity of which is still
unknown. Research on the atractylate-insensitive
site has yielded scarce, moderately conflicting, but
important information. The presence of an additional
ADP-binding component other than adenine nucleotide
translocase (ANT) has been suggested [77,78]. ADP has
also been shown to exert an effect on the permeability
transition by interaction at two binding sites, one that
is carboxyatractyloside-sensitive, most likely ANT, and
another that shows low affinity for adenine nucleotides
but is insensitive to atractylates [79]. In another study,
addition of 2 mm ADP after 5 lm carboxyatractyloside
did not change respiration rates in mouse liver mito-
chondria, but increased mitochondrial Ca
2+
uptake

capacity more than fivefold [80]. In this study, the K
i
for PTP inhibition in Mg
2+
-free medium was estimated
to be 0.9 mm ADP. Using de-energized rat liver mito-
chondria, Halestrap calculated a K
i
for PTP inhibition
as low as 0.025 mm ADP, but this was in the presence
of Mg
2+
in the medium [85]. In another study, Mg
2+
and ADP were found to close the pore in rat liver mito-
chondria in a carboxyatractyloside-insensitive manner
[81]. Furthermore, the presence of a low-affinity ADP-
binding (K
m
77 lm) non-carboxyatractyloside binding
site appeared to confer increased sensitivity to cyclo-
sporin A [82–84]. This low-affinity ADP-binding site
was suggested to reside on the matrix side of the inner
mitochondrial membrane. However, Mg
2+
was impli-
cated in modulation of PTP opening by binding to a site
located on the outer part of the inner mitochondrial
membrane [46]. However, in other studies on heart mito-
chondria, ADP was found to be ineffective [85–87]. In

rat liver mitochondria, an ADP binding site other than
ANT has been reported to confer resistance to PTP
opening, with a K
i
of 0.07 mm [83]. ATP was shown to
delay PTP opening, but only at 0.3 mm concentration
and in the presence of cyclosporin A. In hamster brown
adipose tissue mitochondria, an atractylate-insensitive
site located on the outer face of the inner mitochondrial
membrane with an affinity for purine nucleotides (ADP
and GDP) binding and a capacity of 0.7 nmolÆmg
)1
pro-
tein has been reported [88]. GDP was found to compete
with ADP. The affinity constant was dependent on pH,
that for GDP being 4.2 lm at pH 6.7 and 34 lm at pH
7.9. However, no such site was found in rat liver [88]. In
beef heart and pig heart mitochondria, ADP was found
to inhibit Ca
2+
-induced PTP opening in the presence of
carboxyatractyloside [84], and ADP was more potent
than ATP. The EC
50
for ADP was estimated as 0.09 mm
and that for ATP was estimated as 0.18 mm.
If ATP uptake were a requirement for an increase in
maximum Ca
2+
uptake capacity, ATP hydrolysis by

F
0
F
1
-ATPase (provided that mitochondrial membrane
potential is sufficiently decreased, see below) should
provide inorganic phosphate for formation of precipi-
tates; however, in the presence of ATP, inorganic
phosphate was found not to be absolutely critical for
Ca
2+
uptake [89,90], and the presence of oligomycin
did not alter these outcomes. DeLuca and Engstrom
[90] reported that inorganic phosphate is not necessary
in the presence of ATP, while Vasington and Murphy
[89], using similar conditions, showed that omission of
inorganic phosphate decreased the amount of Ca
2+
taken up by 40–70%. However, experiments investigat-
ing the effect of exogenously added adenine nucleo-
tides on Ca
2+
-phosphate precipitation in the matrix
must be evaluated by electron microscopy, not by
following changes in light scattering. This is because
the inner mitochondrial membrane is known to con-
tract in response to addition of adenine nucleotides, an
effect that is mediated by ANT, creating changes in
mitochondrial optical density, but this is unrelated to
matrix precipitate formation [77,78].

C. Chinopoulos and V. Adam-Vizi Mitochondrial Ca
2+
precipitation
FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS 3641
The above reports indicate that there is a site on
mitochondria other than ANT that increases maximum
Ca
2+
uptake capacity, and that it binds adenine nucle-
otides with an affinity lower than that of ANT. How-
ever, there is no consensus on whether this site is
located on the outer or the inner leaflet of the inner
mitochondrial membrane in mitochondria of the vari-
ous tissue types, whether it binds both ADP (or GDP)
and ATP and with what affinities, and what the role
of Mg
2+
is in this binding, if any. Additionally, there
is still no information regarding the identity of this
atractylate-insensitive adenine nucleotide binding site.
With regard to the uptake of adenine nucleotides
through ANT as a prerequisite for operation of the
atractylate-sensitive site, many reports seem to disre-
gard the influence of DW
m
on the directionality of
ANT [91–93]. ANT operates in forward mode, i.e. it
brings ADP into the matrix in exchange for ATP, if
DW
m

is more negative than $ )100 mV, and works in
reverse, i.e. brings ATP into the matrix in exchange
for ADP, if DW
m
is more positive than $ )100 mV
[47,94–96]. A typical ADP–ATP exchange rate–DW
m
profile for isolated rat liver mitochondria is shown in
Fig. 3A. Mitochondria expel ATP in exchange for
ADP if their membrane potential is in the range from
)145 to )100 mV, but consume extra-mitochondrial
ATP at more positive DW
m
values. The membrane
potential value at which there is no net transfer of
ADP–ATP across the inner mitochondrial membrane
is the reversal potential of ANT (E
rev_ANT
). By ther-
modynamic deduction, E
rev_ANT
is given by:
where ‘out’ indicates outside the matrix, ‘in’ indicates
inside the matrix, R is the universal gas constant
(8.31 JÆmol
)1
ÆK
)1
), F is the Faraday constant
(9.64 · 10

4
CÆmol
)1
) and T is the temperature (in
Kelvin) [94]. There is no doubt that both ADP and
ATP accumulate in mitochondria during Ca
2+
loading
[97], but the question is how to reconcile the entry of
both adenine nucleotide species with the mutually exclu-
sive forward and reverse modes of ANT operation.
To explain this, we show the results of superimposed
time courses of DW
m
and extra-mitochondrial Ca
2+
during multiple additions of CaCl
2
to rat brain mito-
chondria in the presence of 3 mm ATP and 0.8 mm
ADP. It is a widely acknowledged, but, to the best of
our knowledge, unreferenced concept, that measuring
maximum Ca
2+
uptake capacity by monitoring DW
m
using a potential-sensitive fluorescent probe yields
lower values than if a Ca
2+
-sensitive probe is used that

is distributed in the extra-mitochondrial space. There
is only a single report showing that safranine O,
a fluorescent probe that responds to changes in DW
m
,
A
C
B
D
Fig. 3. (A) Plot of the ATP–ADP exchange
rate mediated by ANT versus DW
m
in
isolated rat liver mitochondria depolarized to
various voltages by various amounts of the
uncoupler SF 6847. (B) Combined traces of
DW
m
(gray line) and extra-mitochondrial Ca
2+
(black line) during stepwise additions of
20 l
M CaCl
2
to isolated rat brain mitochon-
dria in high-K
+
ionic strength medium sup-
plemented with 3 m
M ATP, 0.8 mM ADP

and 2 m
M MgCl
2
. (C) Lead-contrasted image
of a mitochondrion visualized by electron
microscopy, sampled after time point ‘c’ as
shown in (B) (magnification: · 50 000). (D)
Lead-contrasted image of a mitochondrion
visualized by electron microscopy, sampled
from the time interval ‘a’–‘b’ as shown in
(B) (magnification · 50 000).
E
rev ANT
¼
2:3RT
F
 log

½ADP
3À

free
out ½ATP
4À

free
in
0
½ADP
3À


free
in ½ATP
4À

free
out
!
Mitochondrial Ca
2+
precipitation C. Chinopoulos and V. Adam-Vizi
3642 FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS
decreases maximum Ca
2+
uptake capacity if used at a
concentration above 5 lm [98]. If safranine O is used
properly, i.e. at 2.5 lm (as for the results shown in
Fig. 3B), it does not affect mitochondrial bioenergetics.
As shown in Fig. 3B, mitochondria accumulate nine
pulses of 20 lm CaCl
2
without any alterations in the
baseline of DW
m
(up to point ‘a’). From point ‘a’ to
point ‘b’, mitochondria accumulate ten additional
equimolar CaCl
2
pulses with an apparently undimin-
ished avidity, but DW

m
starts to become more positive.
The reasons for the gradual decrease in DW
m
upon
excessive Ca
2+
accumulation is beyond the scope of
this review, but may include inhibition of a-ketogluta-
rate dehydrogenase [99] and complex I [100] [101] by
high amounts of Ca
2+
. In the interval between points
‘a’ and ‘b’, DW
m
remains in the range within which
ANT operates in the forward mode, and thus mito-
chondria are able to take up ADP in exchange for
ATP. However, this does not apply for F
0
F
1
-ATPase,
as the reversal potential of this complex (E
rev_ATPase
)is
more negative than that of ANT [94]. By thermody-
namic deduction, E
rev_ATPase
is given by:

and
½P
À

in
¼½P
total

in

1 þ 10
pH
i
ÀpK
a2
ÀÁ
where ‘o’ or ‘out’ indicate outside the matrix, ‘i’ or ‘in’
indicateinside the matrix, n is the H
+
⁄ ATP
coupling ratio, R is the universal gas constant (8.31
JÆmol
)1
ÆK
)1
), F is the Faraday constant (9.64 · 10
4
C
mol
)1

), T is the temperature (in Kelvin), [P
)
] is the free
phosphate concentration in Molar, and pK
a2
= 7.2
for phosphoric acid [94]. The reversal of F
0
F
1
-ATPase
due to a high rate of Ca
2+
uptake has been known for
35 years [102]. However, under the conditions
described above, a paradox emerges in which the activ-
ity of F
0
F
1
-ATPase is reversed, thereby consuming
mitochondrial ATP, but ANT is still operating in the
forward mode [94], and therefore extra-mitochondrial
ATP cannot be provided for the F
0
F
1
-ATPase. Six
additional equimolar Ca
2+

pulses are sequestered by
the mitochondria after point ‘b’ (although the uptake
rate starts to decrease), and after point ‘b’, DW
m
attains values at which ANT also reverses, thereby
allowing extra-mitochondrial ATP to enter the matrix.
At point ‘c’, mitochondria are completely depolarized,
although they are still able to remove extra-mitochon-
drial Ca
2+
, albeit at a rapidly deteriorating uptake
rate. These results indicate that when mitochondria are
challenged with sufficiently high amounts of Ca
2+
,
DW
m
will eventually decrease to a degree that allows
reversal of ANT and import of extra-mitochondrial
ATP into the matrix. The fate of adenine nucleotides
that have been take up with respect to formation of
the precipitates is discussed below. It is worth empha-
sizing the ability of mitochondria with no measurable
membrane potential to remove extra-mitochondrial
Ca
2+
. Mitochondria sampled for electron microscopic
inspection from such an experiment from point ‘c’
onwards exhibit classic signs of PTP. Many of them
appear as shown in Fig. 3C, broken open but with pre-

cipitates in them. Mitochondria fixed before point ‘b’
and visualized by electron microscopy appear as shown
in Fig. 3D. The property of mitochondria to undergo
permeability transition but retain Ca
2+
-phosphate pre-
cipitates has been reported previously [48]. However,
we propose that broken mitochondria might chelate
exogenously added Ca
2+
, as exogenously added CaCl
2
has unobstructed access to the precipitation machinery
in broken mitochondria. That could account for the
paradox that extra-mitochondrial Ca
2+
is sequestered
by mitochondria with no membrane potential that
exhibit obvious signs of PTP opening (from point ‘c’
until the beginning of Ca
2+
release).
What is the nature of the mitochondrial
precipitates formed upon Ca
2+
uptake?
Precipitation of Ca
2+
during various pathological
conditions has been observed in several intracellular

locations, including mitochondria [10,11,103,104].
Within isolated or in situ mitochondria, the precipi-
tates take the form of granules that almost always
show electron-transparent cores [42], often in associa-
tion with the inner membranes [42]. This pattern is
similar even if the divalent cation is Ba
2+
or Sr
2+
(see Fig. 4A). The electron opacity of the rim of these
granules does not depend upon heavy-metal staining
but is intrinsic [76]. However, in isolated mitochon-
dria from certain tissues (such as rabbit heart) that
sequester large amounts of Ca
2+
in the absence of
Mg
2+
, the precipitates appear as needles instead
(Fig. 4B,C) [105].
The granules formed in the mitochondrial matrix
upon Ca
2+
sequestration contain an inorganic and an
organic moiety [41,76]. Depending on the method of
E
rev ATPase
¼Àð316=nÞÀ

2:3RT

F=n

 log

½ATP
4À

free
in
0
½ADP
3À

free
in ½P
À

in
!
À
2:3RT
F
ÂðpH
o
À pH
i
Þ
C. Chinopoulos and V. Adam-Vizi Mitochondrial Ca
2+
precipitation

FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS 3643
granule isolation, the organic moiety accounts for
16–60% of the total (Fig. 4D). The organic moiety
appears to occupy the electron-transparent core, while
the inorganic moiety is located in the electron-dense
rim. The physico-chemical properties of the granules
imply an intimate association of the organic with the
inorganic constituents [76]. The inorganic moiety is rich
in Ca
2+
,P
i
,Mg
2+
and CO
2À
3
,corresponding to hydroxy-
apatite Ca
10
(PO
4
)
6
(OH)
2
, whitelockite Ca
3
(PO
4

)
2
,or
a mixture of both, as well as traces of MgO, presumably
derived from MgCO
3
[41]. Subsequent studies have
concluded that the hydroxyapatite present in the inor-
ganic moiety of the granules is Ca
2+
-deficient [76].
The composition of the constituents of the inorganic
moiety can be manipulated by the rate of Ca
2+
infusion
to isolated mitochondria [48], and also by endogenous
factors (see below). This may be due to alleviation
against bursts of metabolic compensations, as moni-
tored by alterations in state 4 respiration [25,106]. The
organic moiety contains nitrogen and tests positive in a
biuret test, indicating the presence of protein(s), and
also for ribose, implying the presence of RNA [41].
Most of the P
i
in the inorganic moiety found in
the granules originates from the medium, and only 16%
of the initial specific activity of labeled ATP is found
in the granules [41]. However, during loading of
mitochondria with Ca
2+

, there is an unexplained anion
deficit that cannot be fully accounted for by complex-
ation to phosphate [107]. Complexation of Ca
2+
and its
salts to the organic moiety may account for this anion
deficit.
However, as noted previously [25], there is an addi-
tional puzzle regarding Ca
2+
-phosphate complexation,
namely reconciliation of the apparent properties of
the matrix Ca
2+
-phosphate complex with those of
known complexes in solution. ‘Physiological’ incuba-
tion media for cells contain millimolar [Ca
2+
] in the
presence of millimolar [P
i
]; furthermore, in experi-
ments with isolated mitochondria, CaCl
2
is added in
the submillimolar concentration range in media that
also contain millimolar amounts of P
i
, and yet an
osmotically inactive complex forms in the matrix

when [Ca
2+
]
m
rises above 1–5 lm [25,26]. Why is
there no formation of precipitates outside the matrix?
At least one study has shown precipitate formation
outside the matrix, but it used the pyroantimonate
technique [108], the validity of which is disputed [48].
An obvious conclusion is that ‘the mitochondrial
matrix is perhaps as far from an ideal solution as it
is possible to imagine’ [25]. Perhaps the organic moi-
ety serves the purpose of a ‘scaffold’ upon which
Ca
2+
precipitates with phosphate, given the low con-
centration of the former compared to the latter. If a
protein exists that plays this scaffolding role, it would
be of great value to know its identity.
However, the mystery of Ca
2+
-phosphate precipita-
tion in the mitochondrial matrix is only one side of the
coin. The other is the reason(s) behind the lack of
transition of calcium phosphate deposits to hydroxyap-
atite, best exemplified by the late Albert Lehninger, as
‘why we do not all turn into stone’ [109]. Theories of
bone [110] and teeth [111] calcification that implicated
A
C

B
D
Fig. 4. (A) Smooth muscle cell from a toad
urinary bladder incubated for 6 h in calcium-
free Ringer’s solution containing 2 m
M bar-
ium acetate, showing dense intramitochond-
rial granules, most of which appear hollow
(magnification: · 210 000). Reprinted from
[2] with permission from Rockefeller Univer-
sity Press. (B,C) Rabbit cardiac mitochondria
fixed after active Ca
2+
uptake in the pres-
ence (B) and absence (C) of Mg
2+
. Rep-
rinted from [135]. (D) Effect of incineration
temperature on the fine structure of dense
granule residues isolated from formalde-
hyde-fixed, calcium phosphate-loaded mito-
chondria from rat liver (magnification:
· 63 000). The granule residues are bubble-
like; the granule mass appears to fuse at
high incineration temperature and bubbles
are formed as the organic component vapor-
izes. Reprinted from [76] with permission
from Rockefeller University Press.
Mitochondrial Ca
2+

precipitation C. Chinopoulos and V. Adam-Vizi
3644 FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS
mitochondria enjoyed wide attention until the begin-
ning of the 1980s [109,112–115], when it was realized
that ossification and enamel-forming mechanisms are
separate from the calcification processes that occur
within the mitochondrial matrix [116]. However,
because of these studies, the concept of ‘calcific dis-
eases’ emerged; these included major maladies of our
times, such as arthritis, atherosclerosis, urolithiasis,
calcific valvular sclerosis and tumor calcification [117–
119]. Research on these ‘calcific diseases’ yielded dis-
covery of a factor (originally termed ‘Howard factor’)
with the ability to prevent calcification in buffered
solutions containing Ca
2+
and phosphate, preventing
the formation of the hydroxyapatite lattice [109,120].
This factor, which is normally present in urine, blood,
milk and saliva, is absent from individuals who suffer
from repeated calcium oxalate stones in their kidneys
[109]. Unexpectedly, Becker et al. found that rat liver
mitochondria also contain a substance that inhibits
precipitation of calcium phosphate and its conversion
to hydroxyapatite [109]. Subsequent chromatographic
analysis, mass spectrometry and proton NMR identi-
fied this calcification inhibitor, which present in body
fluids and in the mitochondrial matrix, as phosphoci-
tric acid [120–122]. Phosphocitrate was quickly realized
to be the most potent inhibitor of hydroxyapatite crys-

tal growth [120,123] (Fig. 5A,B). Other naturally
occurring substances found in mitochondria are also
known to inhibit hydroxyapatite formation, such as
inorganic pyrophosphate [124], ATP and Mg
2+
[125],
although these are far less potent than phosphocitrate
[120]. Another endogenous substance that disrupts
hydroxyapatite formation is inorganic polyphosphate.
Polyphosphate is a polymer comprising as few as ten
to several hundred phosphate molecules linked by
ATP-like high-energy bonds, and has been found in all
eukaryotic organisms tested, localized in various com-
partments, including the mitochondria [126]. Polyphos-
phate has strong links to the mitochondrial Ca
2+
sequestration system: (a) it is implicated in composi-
tion of the ion-conducting module of the PTP
[127,128], (b) reduction of polyphosphate levels
increases mitochondrial Ca
2+
uptake capacity and
decreases the probability of pore opening [129], (c) it is
a chelator of Ca
2+
, among other divalent ions [129],
and (d) it inhibits calcium hydroxypatite crystal growth
[130] (Fig. 5C,D). Furthermore, polyphosphate levels
and mitochondrial bioenergetic parameters are recipro-
cally regulated [131,132], and parameters such as the

mitochondrial membrane potential and ATP produc-
tion by the F
0
F
1
-ATPase are important determinants
of mitochondrial Ca
2+
uptake capacity [31]. Phospho-
citrate [133] and polyphosphate occur naturally within
mitochondria, however, it is not known how they
are produced [126]; phosphocitrate can easily be
chemically synthesized [134].
cm
mm
mm
A
B
C
D
Fig. 5. (A,B) Electron microscopic study of the effects of phosphocitrate on parathyroid hormone-induced nephrocalcinosis. Thin sections
from mice treated with phosphocitrate and parathyroid hormone or saline and parathyroid hormone for 4 days were fixed and stained with
uranyl acetate and lead citrate (magnification: · 12 400). The control (saline ⁄ parathyroid hormone) sections (A) contained numerous heavily
mineralized mitochondria (mm) as well as extensive areas of calcification within the cytoplasm (cm). Mineral deposits were not observed in
sections from those animals given phosphocitrate prior to parathyroid hormone (B). Adapted from [122]. (C,D) The appearance of freshly pre-
cipitated calcium orthophosphate in solution (magnification: · 190 000) (C) and freshly precipitated calcium orthophosphate inhibited by poly-
phosphate in solution (magnification: · 190 000) (D). Adapted from [130].
C. Chinopoulos and V. Adam-Vizi Mitochondrial Ca
2+
precipitation

FEBS Journal 277 (2010) 3637–3651 ª 2010 The Authors Journal compilation ª 2010 FEBS 3645
Conclusions
In this review, we have re-examined the thermo-
dynamic aspects of mitochondrial Ca
2+
uptake and
release mechanisms, processes that have been investi-
gated for decades and still generate a vast amount of
literature. The major ‘players’ in Ca
2+
uptake and
release mechanisms are still unknown: the molecular
identities of the uniporter and PTP are unknown, and
the identities of the Na
+
⁄ Ca
2+
exchanger and possi-
bly the Ca
2+
⁄ H
+
antiporter have only been revealed
very recently. The action of uncouplers in induction of
mitochondrial Ca
2+
release also remains inadequately
explained, although it is now accepted that the effect
on matrix acidification accounts for only a fifth of the
total amount of Ca

2+
that can be released. Further-
more, the thermodynamic aspects of the role of
adenine nucleotides in mitochondrial Ca
2+
uptake
capacity and precipitate formation have been exam-
ined, and placed under the perspective of the direc-
tionality of ANT operation. The possible existence of
an atractylate-insensitive, adenine nucleotide binding
site that modulates Ca
2+
uptake capacity is re-
appraised. Finally, the nature of the Ca
2+
-phosphate
precipitates formed in the mitochondrial matrix has
been re-addressed, and it will be insightful to deter-
mine the composition of the organic moiety, and to
resurrect the concepts on the origin and regulation of
the endogenous Ca
2+
-P
i
hydroxyapatite lattice break-
ers, such as phosphocitrate and polyphosphate. The
present review provides more questions than answers,
but the key to fruitful research is to ask the right
question!
Acknowledgements

We thank Dr Akos A. Gerencser, Buck Institute,
Novato, CA, USA for generating the model of the
mitochondrial Na
+
⁄ Ca
2+
exchanger. The work by our
group referred to in the text was supported by grants
from Orsza
´
gos Tudoma
´
nyos Kutata
´
si Alapprogram
(OTKA), Magyar Tudoma
´
nyos Akade
´
mia (MTA),
Nemzeti Kutata
´
si e
´
s Technolo
´
giai Hivatal (NKTH)
and Egeszsegu
¨
gyi Tudoma

´
nyos Tana
´
cs (ETT) to V.A
V., and by OTKA-NKTH grant number NF68294 and
OTKA grant number NNF78905 to C.C.
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