Current Biology 18, 855859, June 3, 2008 2008 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2008.05.026

Ca -Dependent Inactivation of the Mitochondrial Ca2+ Uniporter Involves Proton 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 Whereas the second Ca2+ pulse (Control Peak 2 in Figure 1C) evoked no detectable mitochondrial Ca2+ rise after the prepulse (2 6 1%, p > 0.4; n = 176 cells), a substantial Ca2+ rise occurred when NH4Cl was added between the pulses (peak 2 was now 31 6 6% that of peak 1; p < 0.001; n = 188 cells; Figure 1C). Matrix Ca2+ just prior to the second Ca2+ pulse was not significantly different for the two conditions (F/F0 was 1.21 6 0.10 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 signicant 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 acidication to increase it. To test this, we applied the weak acid sodium acetate (10 mM) to permeabilized cells prior to a 50 mM Ca2+ pulse. Sodium acetate results in prominent matrix acidication [20]. After exposure to acetate, mitochondrial Ca2+ uptake was dramatically reduced (Figure 1D; p < 0.001; 89 cells for control and 77 for acetate). Matrix Ca2+ (F/F0) was not signicantly 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, respectively, 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 acid might reect changes in cytosolic pH rather than in matrix pH. 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 mitochondrial Ca2+ uptake after the second Ca2+ application in the presence of NH4Cl (data not shown). Third, we considered the possibility that rhod-2 might be pH-dependent such that its uorescence increases in an alkaline environment and decreases 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 conrm that this was also the case in our experimental system, we measured rhod-2 uorescence 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 in order 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 inhibitor 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 prevented [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, acidication 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

2+

Report

Summary Stimulation of receptors on the surface of animal cells often evokes cellular responses by raising intracellular Ca2+ concentration [1]. The rise in cytoplasmic Ca2+ drives a plethora of processes, including neurotransmitter release, muscle contraction, and cell growth and proliferation [2]. Mitochondria help shape intracellular Ca2+ signals through their ability to rapidly take up signicant 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 cytoplasmic Ca2+ rise prevents further Ca2+ uptake [15]. In spite of its importance in intracellular Ca2+ signaling, little is known about the mechanism underlying uniporter inactivation. Here, we report that maneuvers that promote matrix alkalinisation signicantly reduce inactivation whereas acidication exacerbates it. We further show that the F1F0ATP synthase complex is an important source of protons for inactivation of the uniporter. These ndings 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 inactivation of the uniporter, we measured Ca2+ concentration within the mitochondrial matrix by using the uorescent Ca2+ indicator rhod-2 in permeabilized RBL-1 cells. Application of 20 mM Ca2+ to the cytosol resulted in a rise in mitochondrial Ca2+ (Figure 1A), which was prevented by (1) inhibition of the uniporter with ruthenium red and (2) depolarization of the inner-membrane potential with the protonophore FCCP [15]. Mitochondrial 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 inactivation of the uptake pathway [15]. Because several Ca2+-selective channels can be gated by protons (H+) [1619], we examined 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 signicantly more mitochondrial Ca2+ uptake when the second Ca2+ pulse (150 mM) was applied (Figure 1B). Aggregate data is summarized 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).

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

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Figure 1. Alteration of Matrix pH Affects Ca2+Dependent Inactivation of the Uniporter Mitochondrial Ca2+ was measured in permeabilized RBL-1 in which rhod-2 had been compartmentalised within the mitochondrial matrix. (A) After a pulse of Ca2+ (prepulse; 20 mM), application of a higher concentration of Ca2+ (150 mM) failed to evoke a mitochondrial Ca2+ rise. A pulse of NH4Cl, applied between the Ca2+ pulses, enabled mitochondrial Ca2+ uptake to occur in response to the second Ca2+ pulse (B). Aggregate data from several such experiments is summarized in panel (C), in which Peak 1 is the peak size of the 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.

alterations in matrix pH could secondarily increase matrix Ca2+ and that it is the latter that might trigger uniporter inactivation. Two arguments can be raised against this possible mechanism. First, the uniporter remained inactive even when mitochondrial 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 acidication after the rst Ca2+ pulse, then one would predict that Ca2+ uptake into mitochondria by ionomycin should be increased once inactivation has developed. Ionomycin is generally considered an electroneutral ionophore that transports Ca2+ in exchange for two protons [23]. Hence, acidication of the matrix should accelerate the ionophores Ca2+-transporting capacity. We compared the rate of rise and extent of the matrix Ca2+ increase following ionomycin application (in the presence of

100 mM Ca2+) between nonstimulated cells (Figure 2A) and those pre-exposed to a 20 mM Ca2+ prepulse in order to inactivate the uniporter (Figure 2B). In both cases, the uniporter was blocked just prior to challenge with ionomycin by application of ruthenium red (100 mM [15]). This enabled us to measure ionomycinmediated 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 Figure 2A), which would reduce the concentration gradient for mitochondrial Ca2+ uptake, the mitochondrial Ca2+ rise evoked by ionomycin was substantially 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 subsequent rise in matrix H+ concentration after uniporter inactivation. We tried to measure matrix pH directly by using the genetically 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 acidication of the matrix on perfusion with 20 mM Ca2+, but this was not always observed. Given that acidication to FCCP was also quite variable, we suspect that the low expression levels of the probe, as well as other ill-dened factors, render the use of this probe in RBL-1 cells somewhat difcult in our hands. How might matrix H+ rise after Ca2+ entry into the matrix? Sources for H+ entry into the matrix include Ca2+-H+ exchange, an ill-dened H+-leak pathway, and H+ entry through the F1F0ATP 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 synthase is extremely important physiologically because the free energy liberated as H+ moves down its electrochemical gradient is harnessed to synthesize ATP. H+ conduction through the

Mitochondrial pH Inactivates Mitochondrial Calcium Uptake 857

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 inhibited by 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, conrming inactivation of the uniporter by the 20 mM Ca2+ prepulse. Aggregate data are presented as mean 6 SEM.

protein can be blocked by oligomycin, and this results in alkalinisation of matrix pH [20]. We therefore examined the effects of oligomycin (0.5 mg/ml) on cytosolic Ca2+-dependent inactivation of the uniporter. Whereas a Ca2+ prepulse evoked almost complete inactivation of the uniporter (Figure 3A, n = 88 cells), pretreatment with oligomycin substantially reduced the extent of inactivation (Figure 3B, n = 74 cells; p < 0.001), pointing to a role for H+ ux through the ATP synthase in uniporter inactivation. Aggregate data are summarized in Figure 3C. Matrix Ca2+ just prior to the second Ca2+ pulse was similar for control cells (F/F0:1.14 6 0.11) and those preexposed 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 permeabilized cells were continuously exposed to 2 mM Mg-ATP. Conclusions Through their ability to rapidly take up cytosolic Ca2+, mitochondria are integral components of the Ca2+-signaling machinery. Mitochondrial Ca2+ buffering not only sculpts the spatial and temporal proles of the cytoplasmic Ca2+ rise (reviewed in [5]) but also stimulates mitochondrial ATP production [24, 25]. In some cell types, the mitochondrial uniporter exhibits inactivation, in that Ca2+ uptake is reduced during prolonged cytosolic Ca2+ rises [12, 13, 15]. It has been clearly established that mitochondria can be exposed to high local Ca2+ emanating from open InsP3 or ryanodine receptors [5] or Ca2+-permeable plasma-membrane channels [26], and these Ca2+ microdomains often exceed 10 mM [15, 27]. Hence, the Ca2+ concentrations that we have used here are relevant physiologically. Ca2+dependent inactivation of the uniporter occurs more slowly than does mitochondrial Ca2+ uptake and has a time constant of w16 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+ can remain elevated long after cytoplasmic Ca2+ has returned to resting levels [29], similar to what we have found here. Uniporter inactivation imparts plasticity to mitochondrial Ca2+ buffering, but how cytosolic Ca2+ inactivates the uniporter is unclear. We now report that Ca2+-dependent inactivation is mediated, at least in part, by alterations in mitochondrial pH. Acidication accelerates inactivation, whereas alkalinisation reduces it. A major source of protons emanate from the ATP synthase, because blocking H+ ux through the enzyme complex reduces the extent of Ca2+-dependent inactivation of the uniporter. It is not clear whether uniporter inactivation requires bulk matrix acidication or whether this is a local phenomenon. Measurements of mitochondrial pH with a genetically targeted green uorescent 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+ release 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 acidication after a cytoplasmic Ca2+ rise has been reported (Poburko and Demaurex, Biophysical Journal Abstract, 2007; personal communication). Similar ndings have been made in cortical neurons, in which glutamate induces a sustained matrix acidication [30]. In view of the high matrix H+-buffering capacity, it is unlikely that cytosolic Ca2+ pulses of 1020 mM, sufcient to inactivate the uniporter [15], would cause large matrix pH uctuations. Intriguingly, protons can move along mitochondrialmembrane 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 membrane surface [31]. Such proton coupling between the F1F0-ATP synthase and the uniporter channel could allow for very effective 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.

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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 rst Ca2+ pulse, and Ctrl peak 2 represents the matrix Ca2+ response to the second 150 mM Ca2+ pulse. Oligo Peaks 1 and 2 are the corresponding values after pretreatment with oligomycin. Aggregate data are presented as mean 6 SEM.

were carried out with the IMAGO charge-coupled-device camera-based system from TILL Photonics. Supplemental Data One supplemental gure 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. USA 99, 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). Mitochondrial calcium transport: Physiological and pathological relevance. 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., Lifshitz, L.S., Tuft, R.A., and Pozzan, T. (1998). Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 17631766. 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. Hoth, M., Fanger, C., and Lewis, R.S. (1997). Mitochondrial regulation of store-operated calcium signaling in T lymphocytes. J. Cell Biol. 137, 633648. Gilabert, J.-A., and Parekh, A.B. (2000). Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca2+ current ICRAC. EMBO J. 19, 64016407. 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. Kirichok, Y., Krapivinsky, G., and Clapham, D.E. (2004). The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427, 360364. Collins, T.J., Lipp, P., Berridge, M.J., and Bootman, M.D. (2001). Mitochondrial calcium uptake depends on the spatial and temporal prole of cytosolic calcium signals. J. Biol. Chem. 276, 2641126420.

Mitochondrial Ca2+ uptake stimulates ATP synthesis through activation of matrix enzymes pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase [5, 24]. ATP synthesis is driven by H+ ow through the ATP synthase into the matrix. The protons then inactivate the 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 intracellular medium designed 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

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