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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: anant.parekh@dpag.ox.ac.uk

mailto:anant.parekh@dpag.ox.ac.ukmailto:anant.parekh@dpag.ox.ac.uk

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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

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