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Current Biology 18, 855859, June 3, 2008 2008 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2008.05.026
ReportCa2+-Dependent Inactivation of theMitochondrial Ca2+ Uniporter InvolvesProton Flux through the ATP Synthase
Ben Moreau1 and Anant B. Parekh1,*1Department of Physiology, Anatomy and Genetics
Oxford University
Parks Road, Oxford OX1 3PT
UK
Summary
Stimulation of receptors on the surface of animal cells often
evokes cellular responses by raising intracellular Ca2+ con-
centration [1]. The rise in cytoplasmic Ca2+ drives a plethora
of processes, including neurotransmitter release, muscle
contraction, and cell growth and proliferation [2]. Mitochon-
dria help shape intracellular Ca2+ signals through their abil-
ity to rapidly take up significant amounts of Ca2+ from the
cytosol via the uniporter [310], a Ca2+-selective ion channel
in the inner mitochondrial membrane [11]. The uniporter is
subject to inactivation [1214], whereby a sustained cyto-
plasmic Ca2+ rise prevents further Ca2+ uptake [15]. In spite
of its importance in intracellular Ca2+ signaling, little is
known about the mechanism underlying uniporter inactiva-
tion. Here, we report that maneuvers that promote matrix
alkalinisation significantly reduce inactivation whereas
acidification exacerbates it. We further show that the F1F0-
ATP synthase complex is an important source of protons
for inactivation of the uniporter. These findings identify
a novel molecular mechanism that regulates the activity of
this ubiquitous intracellular Ca2+ channel, with implications
for intracellular Ca2+ signaling and aerobic ATP production.
Results and Discussion
To study the mechanism underlying Ca2+-dependent inactiva-
tion of the uniporter, we measured Ca2+
concentration within
the mitochondrial matrix by using the fluorescent Ca2+ indica-
tor rhod-2 in permeabilized RBL-1 cells. Application of 20 mM
Ca2+ to the cytosol resulted in a rise in mitochondrial Ca2+ (Fig-
ure 1A), which was prevented by (1) inhibition of the uniporter
with ruthenium red and (2) depolarization of the inner-mem-
brane potential with the protonophore FCCP [15]. Mitochon-
drial Ca2+ recovered gradually, taking approximately 600 s to
recover fully (see also ref [15]). A second Ca2+
pulse evoked
no detectable rise in matrix Ca2+
(Figure 1A), indicating inacti-vationof theuptake pathway [15]. Because several Ca2+-selec-
tive channels can be gated by protons (H+) [1619], we exam-
ined the effects of the altering of matrix pH on uniporter
activity. Matrix alkalinization can be achieved by application
of the weak base NH4Cl [20]. Exposure to 15 mM NH4Cl,
300 s after the initial 20 mM Ca2+ pulse, resulted in significantly
more mitochondrial Ca2+ uptake when the second Ca2+ pulse
(150 mM) was applied (Figure 1B). Aggregate data is summa-
rized in Figure 1C, in which Peak 1 and Peak 2 refer to the
size of the mitochondrial Ca2+ rise during the prepulse (peak
1) and after the subsequent Ca2+ pulse of 150 mM (peak 2).
Whereas the second Ca2+
pulse (Control Peak 2 in Figure 1C)evoked no detectable mitochondrial Ca2+ rise after the pre-
pulse (26 1%, p > 0.4; n = 176 cells), a substantial Ca2+ rise oc-
curredwhen NH4Cl wasaddedbetween thepulses (peak 2 was
now 3166%that ofpeak 1;p < 0.001;n = 188 cells; Figure1C).
Matrix Ca2+ just prior to the second Ca2+ pulse was not signif-
icantlydifferent forthe twoconditions (F/F0 was1.2160.10 for
control and 1.12 6 0.10 in NH4Cl, p > 0.1). Similar results were
obtained when we used another weak base, TMA-Cl. Again,
whereas a second Ca2+ pulse (150 mM) triggered no detectable
Ca2+ rise after the 20 mM Ca2+ pulse (3 6 0.7%; p > 0.36; 56
cells), a significant increase (28 6 4%; p < 0.001; n = 69) was
seen when 15 mM TMA-Cl was applied between the pulses.
If matrix alkalinization decreases uniporter inactivation, one
might expect acidification to increase it. To test this, we ap-
plied the weak acid sodium acetate (10 mM) to permeabilized
cells prior to a 50 mM Ca2+ pulse. Sodium acetate results in
prominent matrix acidification [20]. After exposure to acetate,
mitochondrial Ca2+ uptake was dramatically reduced (Fig-
ure 1D; p < 0.001; 89 cells for control and 77 for acetate). Matrix
Ca2+ (F/F0) was not significantly different between control and
acetate-treated cells just prior to the second Ca2+ pulse.
We considered various other explanations for these results.
First, NH4Cl and acetic acid pulses could hyperpolarize and
depolarize the mitochondrial membrane potential, respec-
tively, thereby altering the electrical gradient for Ca2+ entry.
However, measurement of matrix potential with JC-1 failed
to reveal any clear change in potential to either stimulus
(data not shown). Second, the effects of NH4Cl and acetic
acidmight reflect changes in cytosolic pH ratherthan in matrixpH. However, cytosolic pH was strongly buffered with 10 mM
HEPES in all experiments. Moreover, the raising of cytosolic
HEPES to 30 mM failed to prevent the increase in mitochon-
drial Ca2+ uptake after the second Ca2+ application in the pres-
ence of NH4Cl (data not shown). Third, we considered the
possibility that rhod-2 might be pH-dependent such that its
fluorescence increases in an alkaline environment and de-
creases in an acidic one. However, a careful study by Collins
et al. reported that rhod-2 was independent of pH over a broad
range (pH 6.88 [12]). Nevertheless, to confirm that this was
also the case in our experimental system, we measured
rhod-2 fluorescence after a cytoplasmic Ca2+ rise before and
then after application of NH4Cl. Intact cells were loaded with
rhod-2 and then treated with antimycin A and oligomycin inorder to eliminate Ca2+ signals from dye compartmentalized
within mitochondria. Thapsigargin, an inhibitor of the SERCA
pumps on intracellular stores, was then applied in Ca2+-free
solution together with the plasma-membrane Ca2+ATPase in-
hibitor La3+ (1 mM). Under these conditions, the cytoplasmic
Ca2+ rise due to block of SERCA pumps is sustained because
Ca2+ removal by mitochondria as well as Ca2+ATPases on the
plasma membrane and endoplasmic reticulum has been pre-
vented [21]. After a sustained Ca2+ rise, application of 15 mM
NH4Cl had little effect on the Ca2+ signal measured with rhod
2 (Figure S1), consistent with the previous report by Collins
et al. [12]. Fourthly, acidification of matrix pH can reduce the
solubility product of Ca3(PO4)2, liberating free Ca2+ within the
matrix [22]. We therefore considered the possibility that*Correspondence: [email protected]
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alterations in matrix pH could secondarily increase matrixCa2+
and that it is the latter that might trigger uniporter inactivation.
Two arguments can be raised against this possible mecha-
nism. First, the uniporter remained inactive even when mito-
chondrial Ca2+
had returned to prestimulation levels [15].Second, acute application of 15 mM NH4Cl or 10 mM acetate
did not change matrix Ca2+ detectably (Figure 1B and data
not shown), indicating a weak effect on Ca3(PO4)2 solubility
under our conditions. Therefore, matrix Ca2+ does not seem
to directly govern the extent of inactivation.
If inactivation of the uniporter involves matrix acidification
after the first Ca2+ pulse, then one would predict that Ca2+ up-
take into mitochondria by ionomycinshould be increased once
inactivation has developed. Ionomycin is generally considered
an electroneutral ionophore that transports Ca2+ in exchange
for two protons [23]. Hence, acidification of the matrix should
accelerate the ionophores Ca2+-transporting capacity. We
compared the rate of rise and extent of the matrix Ca2+
in-
crease following ionomycin application (in the presence of
Figure 1. Alteration of Matrix pH Affects Ca2+-
Dependent Inactivation of the Uniporter
Mitochondrial Ca2+ was measured in permeabi-
lized RBL-1 in which rhod-2 had been compart-
mentalised within the mitochondrial matrix. (A)
After a pulse of Ca2+ (prepulse; 20 mM), applica-
tion of a higher concentration of Ca2+ (150 mM)
failed to evoke a mitochondrial Ca2+
rise. A pulseof NH4Cl, applied between the Ca
2+ pulses,
enabled mitochondrial Ca2+ uptaketo occurin re-
sponse to the second Ca2+ pulse (B). Aggregate
data from several such experiments is summa-
rized in panel (C), in which Peak 1 is the peak
size ofthe rhod-2 signal during the Ca2+ prepulse
and Peak 2 refers to the subsequent signal in 150
mM Ca2+. (D) Whereas a 50 mM cytoplasmic Ca2+
pulse evoked a mitochondrial Ca2+ rise (white
bar), pretreatment with 10 mM acetate prevented
this rise from occurring (black bar).
Aggregate data are presented as mean 6 SEM.
100 mM Ca2+
) between nonstimulated
cells (Figure 2A) and those pre-exposed
toa20mM Ca2+ prepulse in order to inac-
tivate the uniporter (Figure 2B). In both
cases, the uniporter was blocked just
prior to challenge with ionomycin by ap-
plication of ruthenium red (100 mM [15]).
This enabled us to measure ionomycin-
mediated mitochondrial Ca2+
uptake in
the absence of a functional uniporter
channel. Although matrix Ca2+ was
slightly elevated following the Ca2+
prepulse (compare Figure 2B with Fig-
ure 2A), which would reduce the con-
centration gradient for mitochondrialCa2+ uptake, the mitochondrial Ca2+
rise evoked by ionomycin was substan-
tially larger and developed more quickly
in those cells in which the uniporter had
been inactivated by the Ca2+ prepulse
(mean responses are superimposed in
Figure 2C). This is consistent with a sub-
sequent rise in matrix H+ concentration
after uniporter inactivation.
We tried to measure matrix pH directly by using the geneti-
cally targeted pH probe elegantly developed by Pozzan and
colleagues [20]. Although the probe expressed in RBL-1 cells,
it did so at low levels. In a few favorable recordings, we saw
a small acidification of the matrix on perfusion with 20 mMCa2+, but this was not always observed. Given that acidifica-
tion to FCCP was also quite variable, we suspect that the
low expression levels of the probe, as well as other ill-defined
factors, render the use of this probe in RBL-1 cells somewhat
difficult in our hands.
How might matrix H+ rise after Ca2+ entry into the matrix?
Sources forH+ entry into the matrix include Ca2+-H+ exchange,
an ill-defined H+-leak pathway, and H+ entry through the F1F0-
ATP synthase. This latter enzyme has a stalk (F0 domain),
which is an H+-conducting channel, and an enzymatic F1
domain that is composed of ATP synthase. The F1F0-ATP syn-
thase is extremely important physiologically because the free
energy liberated as H+ moves down its electrochemical gradi-
ent is harnessed to synthesize ATP. H+ conduction through the
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protein can be blocked by oligomycin, and this results in alka-
linisation of matrix pH [20]. We therefore examined the effects
of oligomycin (0.5 mg/ml) on cytosolic Ca2+-dependent inacti-
vation of the uniporter. Whereas a Ca2+
prepulse evoked
almost complete inactivation of the uniporter (Figure 3A,
n = 88 cells), pretreatment with oligomycin substantially re-
duced the extent of inactivation (Figure 3B, n = 74 cells;
p < 0.001), pointing to a role for H+
flux through the ATP syn-
thase in uniporterinactivation. Aggregatedata are summarized
in Figure 3C. Matrix Ca2+ just prior to the second Ca2+ pulsewas similar for control cells (F/F0:1.14 6 0.11) and those pre-
exposed to oligomycin (F/F0:1.10 6 0.09; p > 0.1). The ability
of oligomycin to partially restore mitochondrial Ca2+ uptake
was not due to a loss of cytosolic ATP, because the permeabi-
lized cells were continuously exposed to 2 mM Mg-ATP.
Conclusions
Through their ability to rapidly take up cytosolic Ca2+, mito-
chondria are integral components of the Ca2+-signaling ma-
chinery. Mitochondrial Ca2+ buffering not only sculpts the spa-
tialand temporal profiles ofthe cytoplasmic Ca2+ rise(reviewed
in [5]) but also stimulates mitochondrial ATP production [24,
25]. In some cell types, the mitochondrial uniporter exhibitsinactivation, in that Ca2+ uptake is reduced during prolonged
cytosolic Ca2+ rises [12, 13, 15]. It has been clearly established
that mitochondria canbe exposed to high local Ca2+ emanating
from open InsP3 or ryanodine receptors [5] or Ca2+-permeable
plasma-membrane channels [26], and these Ca2+ microdo-
mains often exceed 10mM [15, 27]. Hence, the Ca2+ concentra-
tions that we have used here arerelevantphysiologically. Ca2+-
dependent inactivation of the uniporter occurs more slowly
than does mitochondrial Ca2+
uptake and has a time constant
ofw16 s when exposed to 10 mM Ca2+. Hence, in cell types in
which cytoplasmic Ca2+ oscillates rapidly and mitochondrial
Ca2+ uptake is transientas occurs in cardiac myocytes, for
exampleuniporter inactivation does not develop [28]. On
the other hand, in acutely isolated pancreatic acinar cells,
mitochondrial Ca2+ canremain elevated long after cytoplasmic
Ca2+ has returned to resting levels [29], similar to what wehave
found here.
Uniporter inactivation imparts plasticity to mitochondrial
Ca2+ buffering, but how cytosolic Ca2+ inactivates the uni-
porter is unclear.We nowreport that Ca2+-dependent inactiva-
tion is mediated, at least in part, by alterations in mitochondrial
pH. Acidification accelerates inactivation, whereas alkalinisa-
tion reduces it. A major source of protons emanate from the
ATP synthase, because blocking H+ flux through the enzymecomplex reduces the extent of Ca2+-dependent inactivation
of the uniporter.
It is not clear whether uniporter inactivation requires bulk
matrix acidification or whether this is a local phenomenon.
Measurements of mitochondrial pH with a genetically targeted
green fluorescent protein mutant revealed no clear bulk pH
change within the matrix upon stimulation of cell-surface
receptors with histamine [20], an agonist that triggers Ca2+ re-
lease from intracellular stores. On the other hand, with the use
of the same agonist on the same cell line but with a different
genetically targeted pH probe, prominent matrix acidification
after a cytoplasmic Ca2+ rise has been reported (Poburko and
Demaurex, Biophysical Journal Abstract, 2007; personal
communication). Similar findings have been made in corticalneurons, in whichglutamate induces a sustained matrix acidifi-
cation [30]. Inview ofthehighmatrix H+-buffering capacity, it is
unlikely that cytosolic Ca2+ pulses of 1020 mM, sufficient to
inactivate the uniporter[15], would cause large matrix pH fluc-
tuations. Intriguingly, protons can move along mitochondrial-
membrane surfaces much faster than they can exchange with
the bulk matrix phase, and they can therefore migrate between
two membrane proteins without diffusing away from the mem-
brane surface [31]. Such protoncouplingbetweenthe F1F0-ATP
synthase and the uniporter channel could allow for very effec-
tive local interactions between these two critical mitochondrial
membrane proteins without necessitating a bulk pH change.
Finally, our results provide a molecular mechanism whereby
ATP synthesis can regulate mitochondrial Ca2+ dynamics.
Figure 2. Mitochondrial Ca2+ Uptake Triggered by Ionomycin is Increased after Uniporter Inactivation
(A) Ionomycin evokes a rise in matrix Ca2+ when applied to permeabilized cells in the presence of 100 mM Ca2+.
(B) After an inactivating Ca2+ prepulse, application of ionomycin in 100 mM Ca2+ evokes a larger rise in matrix Ca2+.
(C) The averaged traces from experiments as in panels A and B are superimposed to show the difference in Ca2+ uptake after uniporter inactivation. The
dotted trace represents the response to ionomycin after Ca2+
-dependent inactivation of the uniporter. In panels AC, the uniporter had been inhibitedby 100 mM ruthenium red, applied 25 s before 100 mM Ca2+ (panel A) and 60 s before ionomycin (but after 100 mM Ca2+) (panel B). Note the absence of
response to 100 mM cytoplasmic Ca2+ alone, as shown in panel A, demonstrating that the uniporter had been blocked by ruthenium red. Note also that
100 mM cytoplasmic Ca2+ alone failed to elicit a response, shown in panel B, confirming inactivation of the uniporter by the 20 mM Ca2+ prepulse.
Aggregate data are presented as mean 6 SEM.
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Mitochondrial Ca2+ uptake stimulates ATP synthesis through
activation of matrix enzymes pyruvate dehydrogenase,
NAD+-isocitrate dehydrogenase, and 2-oxoglutarate dehydro-
genase [5, 24]. ATP synthesis is driven by H+ flow through the
ATP synthase into the matrix. The protons then inactivatethe uniporter, providing a mechanism whereby ATP synthesis
can be autoregulated. Such a feedback pathway might be
important in prevention of mitochondrial Ca2+ overload, which
can trigger apoptosis.
Experimental Procedures
RBL-1 cells were cultured as previously described [32]. Mitochondria were
loaded with rhod-2 by incubation with Rhod-2-AM as reported previously,
and cytosolic rhod-2 was removed by permeabilization with digitonin [15].
After permeabilization, cells were perfused with an i ntracellular medium de-
signed to energize the mitochondria (120 mM KCl, 10 mM NaCl, 2 mM
KH2PO4, 10 mM HEPES, 1 mM MgCl2, 1 mM succinic acid, 1 mM pyruvic
acid, 50 mM EGTA, 2 mM Mg-ATP [pH 7.4 with KOH]). Rhod-2 was excited
at 540 nm, and emission > 560 nm was collected. All imaging experiments
Figure 3. Inhibition of the F1F0-ATP Synthase with Oligomycin Reduces
the Extent of Uniporter Inactivation
(A and B) Whereas 150 mM cytoplasmic Ca2+ failed to evoke a response
after uniporter inactivation (A), pretreatment with oligomycin (0.5 mg/ml)
resulted in a prominent matrix Ca2+ rise (B).
(C) Aggregate data from several such experiments are summarized.
Control (Ctr) Peak 1 refers to the initial peak amplitude reached during
the firstCa2+
pulse,and Ctrl peak 2 represents the matrix Ca2+
responseto the second 150 mM Ca2+ pulse. Oligo Peaks 1 and 2 are the corre-
sponding values after pretreatment with oligomycin.
Aggregate data are presented as mean 6 SEM.
were carried out with the IMAGOcharge-coupled-device camera-based
system from TILL Photonics.
Supplemental Data
One supplemental figure is available with this paper online at http://
www.current-biology.com/cgi/content/full/18/11/855/DC1/.
Acknowledgments
This work was supported by a Medical Research Programme grant to
A.B.P. and a British Heart Foundation Studentship to B.M.
Received: December 7, 2007
Revised: May 7, 2008
Accepted: May 7, 2008
Published online: May 29, 2008
References
1. Berridge, M.J., Bootman, M.D., and Roderick, H.L. (2003). Calcium
signalling: Dynamics, homeostasis and remodelling. Nat. Rev.
Mol. Cell Biol. 4, 517529.
2. Carafoli, E. (2002). Calcium signalling: A tale for all seasons. Proc.Natl. Acad. Sci. USA99, 11151122.
3. Duchen, M.R. (2000). Mitochondria and Ca2+ in cell physiology and
pathophysiology. Cell Calcium 28, 339348.
4. Gunter, T.E., Gunter, K.K., Sheu, S.S., and Gavin, C.E. (1994). Mito-
chondrial calcium transport: Physiological and pathological rele-
vance. Am. J. Physiol. 267, C313C319.
5. Rizzuto, R., and Pozzan, T. (2006). Microdomains of intracellular
calcium: Molecular determinants and functional consequences.
Physiol. Rev. 86, 369408.
6. Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lif-
shitz, L.S., Tuft, R.A., and Pozzan, T. (1998). Close contacts with
the endoplasmic reticulum as determinants of mitochondrial Ca2+
responses. Science 280, 17631766.
7. Hajnoczky, G., Hager, R., and Thomas, A.P. (1999). Mitochondria
suppress local feedback activation of inositol1,4,5-trisphosphate
receptors by calcium. J. Biol. Chem. 274, 1415714162.
8. Hoth, M., Fanger, C., and Lewis, R.S. (1997).Mitochondrial regulation ofstore-operated calcium signaling in T lymphocytes. J. Cell Biol. 137,
633648.
9. Gilabert, J.-A., and Parekh, A.B. (2000). Respiring mitochondria deter-
mine the pattern of activation and inactivation of the store-operated
Ca2+ current ICRAC. EMBO J. 19, 64016407.
10. Tinel, H., Cancela, J.M., Mogami, H., Gerasimenko, J.V., Gerasimenko,
O.V., Tepikin, A.V., and Petersen, O.H. (1999). Active mitochondria
surrounding the pancreatic acinar granule region prevent spreading of
inositol trisphosphate-evoked local cytosolic Ca2+ signals. EMBO J.
18, 49995008.
11. Kirichok, Y., Krapivinsky, G., and Clapham, D.E. (2004). The mitochon-
drial calcium uniporter is a highly selective ion channel. Nature 427,
360364.
12. Collins, T.J., Lipp, P., Berridge, M.J., and Bootman, M.D. (2001). Mito-
chondrial calcium uptake depends on the spatial and temporal profile
of cytosolic calcium signals. J. Biol. Chem. 276, 2641126420.
Current Biology Vol 18 No 11858
http://www.current-biology.com/cgi/content/full/18/11/855/DC1/http://www.current-biology.com/cgi/content/full/18/11/855/DC1/http://www.current-biology.com/cgi/content/full/18/11/855/DC1/http://www.current-biology.com/cgi/content/full/18/11/855/DC1/ -
7/30/2019 1-s2.0-S0960982208006647-main.pdf
5/5
13. Maechler, P., Kennedy, E.D., Wang, H., and Wollheim, C.B. (1998). De-
sensitization of mitochondrial calcium and insulin secretion responses
in the beta cell. J. Biol. Chem. 273, 2077020778.
14. Pinton, P., Leo, S., Wieckowski, M.R., Di Benedetto, G., and Rizzuto, R.
(2004). Long-term modulation of mitochondrial Ca2+ signals by protein
kinase C isozymes. J. Cell Biol. 165, 223232.
15. Moreau, B., Nelson, C., and Parekh, A.B. (2006). Biphasic regulation of
mitochondrial Ca uptake by cytosolic Ca concentration. Curr. Biol. 16,16721677.
16. Kaibara,M., andKameyama, M.(1988). Inhibitionof thecalcium channel
by intracellular protonsin single ventricular myocytes of the guinea-pig.
J. Physiol. 403, 621640.
17. Kwan, Y.W., and Kass, R.S. (1993). Interactions between H+ and Ca2+
near cardiac L-type calcium channels: Evidence for independent
channel-associated binding sites. Biophys. J. 65, 11881195.
18. Chen, X.H., Bezprozvanny, I., and Tsien, R.W. (1996). Molecular basis of
H+ block of L-type Ca2+ channel. J. Gen. Physiol. 108, 363374.
19. Talavera, K., Janssens, A., Klugbauer, N., Droogmans, G., and Nilius, B.
(2003). Extracellular calcium modulates the effects of protons on gating
and conduction properties of the T-type calcium channel alpha1G
(Cav3.1). J. Gen. Physiol. 121, 511528.
20. Abad, M.F., Di Benedetto,G., Mgalhaes, P.J., Filippin, L.,and Pozzan, T.
(2004). Mitochondrial pH monitored by a new engineered geen fluores-
cent protein mutant. J. Biol. Chem. 279, 1152111529.
21. Chang, W.C., Di Capite, J., Singaravelu, K., Nelson, C., Halse, V., andParekh, A.B. (2008). Local calcium influx through calcium release-acti-
vated calcium (CRAC) channels stimulates production of an intracellular
messenger and an intercellular pro-inflammatory signal. J. Biol. Chem.
283, 46224631.
22. Chalmers, S., and Nicholls, D.G. (2003). the relationship between free
and total calcium concentrations in the matrix of liver and brain mito-
chondria. J. Biol. Chem. 278, 1906219070.
23. Erdahl, W.L., Chapman, C.J., Taylor, R.W., and Pfeiffer, D.R. (1994).
Ca2+ transport properties of ionophore A23187, ionomycin, and
4-BrA23187 in a well defined model system. Biophys. J. 66, 16781693.
24. McCormack, J.G., Halestrap, A.P., and Denton, R.M. (1990). Role of cal-
cium ions in regulation of mammalian intramitochondrial metabolism.
Physiol. Rev. 70, 391425.
25. Jouaville, L.S., Pinton, P., Bastianutto, C., Rutter, G.A., and Rizzuto, R.
(1999). Regulation of mitochondrial ATP synthesis by calcium: Evidence
for a long-term metabolic priming. Proc. Natl. Acad. Sci. USA 96,
1380713812.
26. Parekh, A.B. (2003). Mitochondrial regulation of intracellular calcium
signaling: More than just calcium buffers. News Physiol. Sci. 18,
252256.
27. Neher, E. (1998). Vesicle pools and Ca2+ microdomains: New tools for
understanding their roles in neurotransmitter release. Neuron 20,
389399.
28. Robert, V.,Gurlini,P., Tosello, V.,Nagai, T.,Miyawaki,A., Di Lisa, F.,and
Pozzan, T. (2001). Beat-to-beat oscillations of mitochondrial [Ca2+] in
cardiac cells. EMBO J. 20, 49985007.
29. Park, M.K., Ashby, M.C., Erdemli, G., Petersen, O.H., and Tepikin, A.
(2001). Perinuclear, perigranular and sub-plasmalemmal mitochondria
have distinct functions in the regulation of cellular calcium transport.
EMBO J. 20, 18631874.
30. Bolshakov, A.P., Mikhailova, M.M., Szabadkai, G., Pinelis, V.G., Brusto-
vetsky, N., Rizzuto, R., and khodorov, B.I. (2007). Measurements of
mitochondrial pH in cultured cortical neurons clarify contribution of
mitochondrial pore to the mechanism of glutamate-induced delayed
Ca2+ deregulation. Cell Calcium, in press. Published online November
23, 2007.
31. Heberle, J., Riesle, J., Thiedemann, G., Oesterhelt, D., and Dencher,
N.A. (1994). Proton migration along the membrane surface and retarded
surface to bulk transfer. Nature 370, 379382.
32. Parekh, A.B., Fleig, A., and Penner, R. (1997). The store-operated
calcium current ICRAC: Nonlinear activation by InsP3 and dissociation
from calcium release. Cell 89, 973981.
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