MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter protein, mitochondrial) that has been shown recently to act as a gatekeeper of the MCU at low [Ca2+]c (cytosolic [Ca2+]). In the present study we have investigated in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we have found several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+ uptake was largely increased at a low [Ca2+]c (<2 μM), but it was decreased at a high [Ca2+]c (>4 μM). In the 2–4 μM range a mixed behaviour was observed, where mitochondrial Ca2+ uptake started earlier in the MICU1-silenced cells, but at a lower rate than in the controls. The sensitivity of Ca2+ uptake to Ruthenium Red and Ru360 was similar at both high and low [Ca2+]c, indicating that the same Ca2+ pathway was operating in both cases. The increased Ca2+-uptake rate observed at a [Ca2+]c below 2 μM was transient and became inhibited during Ca2+ entry. Development of this inhibition was slow, requiring 5 min for completion, and was hardly reversible. Therefore MICU1 acts both as a MCU gatekeeper at low [Ca2+]c and as a cofactor necessary to reach the maximum Ca2+-uptake rate at high [Ca2+]c. Moreover, in the absence of MICU1, the MCU becomes sensitive to a slow-developing inhibition that requires prolonged increases in [Ca2+]c in the low micromolar range.
In recent years, a number of new proteins involved in mitochondrial Ca2+ transport have been discovered . MICU1 (Ca2+ uptake protein 1, mitochondrial) was the first one known to be involved in the mitochondrial Ca2+ uniporter complex . This complex, the main channel responsible for Ca2+ entry into mitochondria, has been found to interact with the MCU (Ca2+ uniporter protein, mitochondrial) protein, which appears to be the main constituent of the pore across the inner mitochondrial membrane [3–5], and with other regulatory proteins such as MCUR1 (mitochondrial Ca2+ uniporter regulator 1)  and MICU2 . Therefore, as could be expected, mitochondrial Ca2+ uptake appears to be a highly regulated process.
The role of MICU1 in the regulation of the mitochondrial Ca2+ uniporter is still unknown. It was discovered in a search for mitochondrial proteins whose knockdown in HeLa cells largely reduced mitochondrial Ca2+ uptake , proving that it was an essential component of the Ca2+-uptake machinery. Given that its predicted structure included only a single membrane-spanning region, it was proposed that it was not the pore, but a regulatory protein essential for transport. However, it has been reported recently that MICU1 is in fact a gatekeeper that limits MCU-mediated Ca2+ influx in order to prevent [Ca2+]M (mitochondrial [Ca2+]) overload . In agreement with this, the main effect of the absence of MICU1 would be to reduce the Ca2+ threshold for the activation of mitochondrial Ca2+ uptake and the effect on the kinetic properties of MCU-mediated Ca2+ uptake would be limited. This view is still under debate, as it has been very recently shown that MICU1 also plays an important role in the co-operative activation of the MCU at a high cytosolic [Ca2+] .
In the present study we have investigated in detail the effect of silencing MICU1 on the kinetics of mitochondrial Ca2+ uptake in HeLa cells. Our results confirm that the absence of MICU1 largely activates mitochondrial Ca2+ uptake at a low cytosolic [Ca2+], but also show that the absence of MICU1 reduces Ca2+ uptake at a higher [Ca2+]c (cytosolic [Ca2+]). In addition, we show that the absence of MICU1 reveals a new mode of functioning of the uniporter at a low cytosolic [Ca2+], where the increased Ca2+ entry induced by MICU1 knockdown is slowly inactivated in the presence of a [Ca2+]c maintained above 200 nM.
MATERIALS AND METHODS
Cell culture, MICU1 knockdown and aequorin expression
HeLa cells stocks are kept frozen in liquid nitrogen. A new stock is opened for use every 2–3 months, and as soon as possible several new samples are frozen again to keep the stock of frozen cells with the minimum passage number. Using this procedure we calculate that our HeLa cells should have no more than 6–8 extra passages since they were originally obtained. Both the morphological and functional characteristics of the cells in terms of Ca2+ signalling have not changed significantly within that time. HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 5% FBS, 100 i.u./ml penicillin and 100 i.u./ml streptomycin. The constructs for mitochondrially targeted mutated aequorin (D119A) and double-mutated aequorin (D119A and N28L) have been described previously [10,11]. Human shRNA constructs made to silence the MICU1 (CBARA1) gene were obtained from Origene (catalogue number TF314182). The most effective was found to be TF314182C (5′-TGCAGAATCTCCACCATGTGTAGACAACC-3′), which was used for most of the present study. The constructs also included the RFP gene to control expression, and were used both for transient transfections and to make stable silenced clones using puromycin by following the manufacturer's instructions. Transfections were carried out using the Metafectene® reagent (Biontex). Preparation of an MCU-silenced HeLa cell clone was described previously .
The experiments described in the present paper were performed after 48–72 h of transfection of this shRNA in HeLa cells (shRNA cells), and compared with controls transfected in parallel with a scrambled shRNA (scRNA cells). In addition, HeLa cell clones expressing either the MICU1-specific shRNA or scrambled shRNA were also obtained and used in some of the experiments. To measure the [Ca2+]M in the transiently transfected cells, the plasmids containing either single- or double-mutated aequorin were co-transfected with the shRNA construct. Cells co-transfected with a scrambled shRNA were used as a control. Measurements were carried out at 48–72 h after transfection. In the case of the stable silenced clone, cells were transfected with the corresponding mitochondrial aequorin construct, and a stable clone expressing a scrambled shRNA cassette was used as a control.
Stable clones were also used to measure the degree of MICU1 knockdown by Western blotting using a polyclonal anti-CBARA1 antibody (Abnova) to recognize MICU1 in the cell extracts. Protein extracts were made from stable clones using RIPA buffer for cell lysis. Proteins were separated by SDS/PAGE (12% gels; Bio-Rad Laboratories), transferred on to PVDF membranes and tested with the polyclonal anti-CBARA1 antibody (1:500 dilution). A monoclonal anti-actin antibody (1:10000 dilution; BD Sciences) was used as the loading control, and was added in a separate lane to avoid overlap with the MICU1 band. Isotype-matched HRP (horseradish peroxidase)-conjugated antibody (1:10000 dilution; Amersham) was used as the secondary antibody, followed by detection by chemiluminescence (Bio-Rad Laboratories). The degree of knockdown in the transiently transfected cells was more difficult to estimate due to uncertainties in the percentage of transfection. The pattern of dynamic [Ca2+]M data obtained in the MICU1-knockdown cell clones was similar to that found in the transient transfections. However, the magnitude of the changes in the [Ca2+]M dynamics induced by MICU1 knockdown was stronger in the transient transfections than in the stable cell clones, suggesting that the percentage of silencing in the transiently transfected cells was probably higher. In addition, there was no difference in the functional response obtained after either 48 or 72 h of transfection, indicating that turnover of MICU1 was very high and the short-term knockdown of this protein was very efficient.
[Ca2+]M measurements with aequorin
HeLa cells were plated on to 13-mm round coverslips before transfection. For aequorin reconstitution, HeLa cells were incubated for 1–2 h at room temperature (20°C) in standard medium [145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose and 10 mM Hepes (pH 7.4)] with 1 μM native coelenterazine (for both single- and double-mutated aequorin; see ). Most of the experiments were performed using mutated aequorin, except for those measuring very high [Ca2+]M values that required use of double-mutated aequorin. After reconstitution, cells were placed in a perfusion chamber of a purpose-built luminometer. For experiments in intact cells, the cells were then perfused with standard medium containing 1 mM [Ca2+] followed by the stimuli. For experiments with permeabilized cells, standard medium containing 0.5 mM EGTA, instead of Ca2+, was perfused for 1 min, followed by 1 min of intracellular medium [130 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM potassium phosphate, 0.5 mM EGTA, 1 mM ATP, 20 μM ADP, 5 mM L-malate, 5 mM glutamate, 5 mM succinate and 20 mM Hepes (pH 7)] containing 100 μM digitonin. The intracellular medium without digitonin was then perfused for 5 min, followed by solutions of known [Ca2+] obtained using either EGTA/Ca2+ or HEDTA (N-hydroxyethylethylenediaminetriacetic acid)/Ca2+/Mg2+ mixtures. The temperature was set at 37°C. Calibration of the luminescence data to the [Ca2+] value was performed using an algorithm adjusted to the calibration of each aequorin form as described previously [12,13]. Statistics analysis was performed by ANOVA.
Measurements of mitochondrial membrane potential
The mitochondrial membrane potential was monitored using the fluorescent indicator TMRE (tetramethylrhodamine ethyl ester). HeLa cells were placed in a cell chamber under a Zeiss Axiovert 200 microscope with continuous perfusion, permeabilized as described above and then perfused with intracellular medium containing 20 nM TMRE until a steady-state fluorescence was reached (usually approximately 5 min). Single-cell fluorescence was excited at 540 nm using a Cairn monochromator and images of the fluorescence emitted between 570 and 630 nm obtained using a ×40 Fluor objective were recorded by a Roper CoolSnap fx camera. Single-cell fluorescence records were analysed off-line using the Imaging Workbench 6.0 program. Experiments were performed at 37°C using an on-line heater from Harvard Apparatus.
Native coelenterazine was obtained from Biotium. Other reagents were from Sigma or Merck.
Silencing of MICU1 has been reported to increase mitochondrial Ca2+ uptake at a low [Ca2+] . In contrast, Ca2+ uptake was either unmodified  or decreased [2,9] when high [Ca2+] was present at the cytosolic side of MICU1-depleted mitochondria. In the present study we have targeted aequorin to investigate in detail the effect of MICU1 knockdown on mitochondrial Ca2+ uptake, and we find several new surprising features of the role for this protein in mitochondrial Ca2+ uniporter activation.
Figure 1 shows the effect of MICU1 knockdown on the [Ca2+]M peak induced by histamine. When the maximum concentration of histamine was used (Figure 1a), MICU1 knockdown reduced significantly the mitochondrial [Ca2+] peak with respect to that obtained in cells transfected with the scrambled shRNA (scRNA cells) (Figure 1e). The same effect was observed at 5 μM histamine (Figure 1b). However, when the cells were stimulated with only 2 μM histamine the [Ca2+]M peak had the same amplitude in MICU1-knockdown cells (shRNA cells) as the controls, and the increase in [Ca2+]M started earlier in the shRNA cells (Figure 1c). Finally, when 0.5 μM histamine was used, a much higher [Ca2+]M increase was obtained in the shRNA cells than in the scRNA cells (Figure 1d). Therefore MICU1 silencing promotes an earlier activation with an increased rate of mitochondrial Ca2+ uptake after low-intensity stimuli, whereas the opposite occurs in the presence of a high-intensity stimuli. Figure 1(e) shows the values of the heights of the Ca2+ peaks induced by each histamine concentration, and Figure 1(f) shows the degree of silencing (approximately 50%) obtained in a cell clone expressing the same shRNA compared with a cell clone expressing a scrambled shRNA. As mentioned in the Materials and methods section, the degree of silencing is probably higher in transient transfections.
Effect of MICU1 knockdown on the mitochondrial [Ca2+] peak induced by different concentrations of histamine
The resting [Ca2+]M has both been reported to be increased significantly  and remain unaltered [2,9] in MICU1-silenced cells. In the present study, we did not find a significant difference. The resting [Ca2+]M was 128±9 nM (mean±S.E.M., n=7) in the scRNA cells and 127±12nM (mean±S.E.M., n=7) in the shRNA cells.
Permeabilized cells were then used to study the mitochondrial Ca2+-uptake process in more detail. Figure 2 shows the effect of MICU1 knockdown on the mitochondrial Ca2+ uptake induced by perfusion of a series of different [Ca2+] buffers in permeabilized cells. After permeabilization, cells were initially perfused with 0.5 mM EGTA-containing medium to ensure that the [Ca2+] was below 10 nM. Perfusion of 100 nM [Ca2+] induced an increase in [Ca2+]M in shRNA cells (Figure 2a), but had no effect in the scRNA cells. In this experiment the [Ca2+]M reached a new higher steady-state in the silenced cells and remained stable there. When 200 nM [Ca2+] was perfused (Figure 2b), there was again an increase in [Ca2+]M in the shRNA cells, but also a very small response in the scRNA cells. In this case, the increase in [Ca2+]M in the silenced cells showed a transient high peak and then decreased to reach a new steady-state. Perfusion of 500 nM [Ca2+] (Figure 2c) also induced only a very small increase in [Ca2+]M in the scRNA cells and a much larger increase in the shRNA cells. At this concentration the transient high peak was also much higher, reaching more than twice the final steady-state concentration. Figure 2(d) shows the effect of perfusion of 1.5 μM [Ca2+] buffer. This concentration was able to trigger a slow, but persistent, increase in [Ca2+]M in the scRNA cells and a very fast, but transient, increase in the shRNA ones.
Effect of MICU1 knockdown on the rate of mitochondrial Ca2+ uptake in permeabilized cells in the presence of different [Ca2+]
When the Ca2+ concentration in the perfusion buffer was 2.5 μM (Figure 2e), the initial increase in [Ca2+]M was still much faster in the shRNA cells, but this increase was short-lived. In contrast the scRNA cells showed an increasing [Ca2+]M after a significant delay, but reached a higher final [Ca2+]M value. Figure 2(e) also shows a trace of the [Ca2+]M increase induced in scRNA cells by 2 μM [Ca2+]. This Ca2+ concentration induces an increase of [Ca2+]M in scRNA cells of a similar magnitude to that induced by 1.5 μM [Ca2+] in shRNA cells, but without the Ca2+-dependent inhibition, suggesting that this effect actually requires MICU1 silencing. Similar findings were obtained at higher [Ca2+] values in the perfusion buffer. At 3.5μM [Ca2+] (Figure 2f), it is still quite evident that the [Ca2+]M increases much earlier in the shRNA cells. In contrast, the increase in [Ca2+]M value in the scRNA cells starts later, but increases at a higher rate and rapidly increases above the levels reached in the shRNA cells. At 4.5 and 10 μM [Ca2+], the rate of increase in [Ca2+]M is larger in the scRNA cells compared with the shRNA cells (Figures 2g and 2h).
Figure 3(a) shows the dependence of the rate of [Ca2+]M increase on the [Ca2+]c for both scRNA and shRNA cells. The Figure shows clearly that silencing of MICU1 increases the rate of mitochondrial Ca2+ uptake only at [Ca2+]c below 2.5 μM. In contrast, for higher [Ca2+]c values the silencing of MICU1 reduces the rate of [Ca2+]M uptake. Figure 3(b) shows that MICU1-silenced cells responded faster to the addition of Ca2+. The delays represent the time from the opening of the perfusion electrovalve to the start of [Ca2+]M increase, and correspond mainly to the time required for the Ca2+ buffer solution to reach the cell coverslip. However, although the same perfusion pathway and electrovalve was used in all the cases, the delays were significantly lower in the shRNA cells compared with the scRNA cells for [Ca2+] values below 2.5 μM.
Effect of MICU1 knockdown on mitochondrial Ca2+ uptake
The lack of difference in the resting [Ca2+]M between the scRNA and shRNA cells appears to be incongruous with the signi-ficant [Ca2+]M increase induced by the addition of 100 nM [Ca2+] in shRNA cells, but not in scRNA cells, shown in Figure 2(a). We have therefore studied if this increase actually leads to a new higher steady-state. Figure 3(c) shows that this is not the case. After the initial increase, the [Ca2+]M value slowly returned to a level close to that of the resting value.
To explore some possible mechanisms for the Ca2+-dependent inhibition, we have performed experiments in the absence of ATP. Perfusing medium without ATP should rapidly wash away cytosolic ATP and lead to the depletion of mitochondrial ATP through ATP/ADP and ATP/Pi exchangers. If the mechanism of inhibition involves any phosphorylation then it should be reduced under these conditions. However, Figure 3(d) shows that the inhibition persisted unchanged. An alternative possibility would be the involvement of ROS (reactive oxygen species) in this phenomenon. We have therefore tested the dynamics of the Ca2+-dependent inactivation in the presence of four different types of antioxidants, both lipid soluble (100 μM α-tocopherol and 100 μM lipoic acid) and water soluble (1 mM ascorbic acid and 5 mM N-methyl-cysteine). None of these antioxidants had any effect on the level of inactivation (Supplementary Figure S1 at http://www.biochemj.org/bj/458/bj4580033add.htm), suggesting that the generation of ROS is not involved.
To be sure that the Ca2+ uptake we have measured above occurs in the mitochondria and takes place through the Ca2+ uniporter, we have tested the effect of the protonophore FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the uniporter inhibitors Ruthenium Red and Ru360. As shown in Figures 4(a) and 4(b), FCCP completely abolished mitochondrial Ca2+ uptake both in shRNA and scRNA cells, indicating that mitochondrial membrane potential is required for the [Ca2+] increase. In addition, we have investigated the inhibition by Ruthenium Red and Ru360 of mitochondrial Ca2+ uptake induced by both high (10 μM) and low (1.5 μM) [Ca2+] in the perfusion buffer, to obtain evidence that it occurred through the same pathway. Our results show that both inhibitors fully blocked mitochondrial Ca2+ uptake, no matter if it was induced by high (Figures 4c and 4e) or low (Figures 4d and 4f) [Ca2+] in the perfusion buffer. Finally, mitochondrial Ca2+ uptake under the same conditions disappeared when a cell clone with silenced MCU was used (Figures 4g and 4h). Thus we can conclude that the Ca2+ uptake measured actually occurred in mitochondria and through the MCU at all of the tested [Ca2+] values.
Effects of FCCP, Ruthenium Red, Ru360 and MCU silencing on mitochondrial Ca2+ uptake in both scrambled shRNA and MICU1-specific shRNA cells
We next studied the mechanism of the peculiar dynamics of [Ca2+]M increase obtained in shRNA cells perfused with low [Ca2+]. As shown in Figure 2, the addition of Ca2+ to the shRNA cells induced a transient increase in [Ca2+]M, which then rapidly decreased to reach a much lower final steady-state. In contrast, in the scRNA cells only a smooth increase in [Ca2+]M was observed at all concentrations of perfused [Ca2+] buffer. This transient high peak suggests the presence of a time-dependent inhibition of the Ca2+ pathway in the presence of the [Ca2+] buffer. To test this possibility, we have designed a protocol with two consecutive perfusions of Ca2+ buffers: the first with a variable [Ca2+] (from 100–500 nM) and the second 5 min later with 1.5 μM [Ca2+]. If the first addition leads to an inhibition of Ca2+ transport through the uniporter, the high transient peak in the second addition should be significantly decreased with respect to that found with single addition of 1.5 μM [Ca2+]. The experimental results confirmed this and the effect was found to be highly dependent on the [Ca2+] in the [Ca2+] buffer of the first perfusion. Figure 5 shows that, when the [Ca2+] in that buffer was 100 nM, there was no effect observed following the subsequent addition of Ca2+. However, when the [Ca2+] in the buffer was 500 nM, the high transient peak following the subsequent addition of 1.5 μM [Ca2+] disappeared. When the first perfusion used a 200 nM [Ca2+] buffer, the inhibition of the high transient peak after the second perfusion was partial; it was still present, but smaller. Therefore our data suggest that the first [Ca2+] buffer perfusion, in the 200–500 nM range, somehow induces an inhibition of the uniporter. The reduction in the second peak induced by previous perfusion of the 500 nM [Ca2+] buffer was 100%, the reduction induced by perfusion of 100 nM [Ca2+] was 24±12% (mean±S.E.M., n=3) and that induced by perfusion of 200 nM [Ca2+] was 78±3% (mean±S.E.M., n=3).
Ca2+-dependent autoinhibition of the mitochondrial Ca2+ uptake activated by MICU1 knockdown
To obtain more information on the mechanism of the inhibition, we studied its time dependence. Figure 6 shows how the inhibition develops progressively when we increase the time of the first Ca2+ perfusion. When the 500 nM [Ca2+] buffer was perfused for only 15 or 30 s (Figures 6c and 6d), there was hardly any effect following the addition of the 500 nM [Ca2+] buffer 5 min later. It produced a [Ca2+]M high transient peak similar to that found when it was perfused with no previous addition of [Ca2+] buffer. As the time of the first perfusion of 500 nM [Ca2+] was increased to 1–2 min (Figures 6b and 6c), the inhibition of [Ca2+]M increase following the second perfusion progressively developed. The time required for 50% inhibition was approximately 2 min (Figure 6f), and the inhibition increased progressively up to 80–90% inhibition after 5–10 min of exposure to the 500 nM [Ca2+] buffer (Figure 6f). Figure 6(f) shows the time course of the inhibition of the second peak. The long length of time required for the inhibition suggests that Ca2+ has to enter into the mitochondria via a mechanism that requires a certain amount of time, because inhibition develops well after the peak of the [Ca2+]M high transient peak. In addition, once produced the inhibition was very long-lasting. In fact, increasing the time interval in EGTA medium between the two 500 nM [Ca2+] additions up to 15 min did not modify the response to the second [Ca2+] addition (results not shown). A partial inhibitory effect was also present when a higher [Ca2+] was perfused in the second addition. As shown in Figure 6(g), the [Ca2+]M increase induced by a 3.5 μM [Ca2+] buffer was reduced by 35% if the 500 nM [Ca2+] buffer had been perfused previously. The inhibition in all of these cases was not due to mitochondrial depolarization, as measurement of mitochondrial membrane potential with TMRE did not detect any change after perfusion of the 500 nM [Ca2+] buffer (Figure 6h).
Time course of the autoinhibition of the mitochondrial Ca2+ uptake activated by MICU1 knockdown
We have also tested the effect of a 5 min perfusion of the 500 nM [Ca2+] buffer on the change in [Ca2+]M following a later [Ca2+] addition in scRNA cells. As shown above, the 500 nM [Ca2+] buffer induces only a small increase in [Ca2+]M in these cells. As shown in Figure 6(i), the second perfusion of 500 nM [Ca2+] in these cells produced a [Ca2+]M increase of similar magnitude, although at a slightly lower rate. When a 3.5 μM [Ca2+] buffer was used as the second perfusion, the increase in [Ca2+]M was slightly, but not significantly, smaller than in the controls (Figure 6j). Thus perfusion of the 500 nM [Ca2+] buffer produces little inhibition in the presence of MICU1.
Since the recent discovery of the molecular substrate of the mitochondrial Ca2+ uniporter, the MCU protein [3,4], a number of other proteins able to modulate Ca2+ transport through MCU have been reported, including MICU2 , MCUR1  and, in particular, MICU1 , which was discovered before MCU and was the first protein found to be related to mitochondrial Ca2+ transport. With few exceptions, MCU and MICU1 are either both present or both absent across all major branches of eukaryotic life . They are also both present in most tissues, although with a variable relationship between them in terms of mRNA expression .
MICU1 has a particularly important role in the regulation of Ca2+ flux through MCU stimulated by low submicromolar [Ca2+]c. Mallilankaraman et al.  found that silencing of MICU1 largely activates mitochondrial Ca2+ uptake at low [Ca2+]c, but has no effect at high cytosolic [Ca2+]. They concluded that it behaves as a gatekeeper of MCU, blocking Ca2+ entry into mitochondria during [Ca2+]c increases of low magnitude. More recently, Csordás et al.  found that MICU1 not only controls the threshold for activation of MCU by [Ca2+]c, but that it also plays an important role in the co-operative activation of MCU by higher levels of [Ca2+]c. Therefore in the absence of MICU1 the rate of Ca2+ uptake by mitochondria at high [Ca2+]c is significantly lower. The results of the present study confirm this double role for MICU1, both as a gatekeeper of MCU at low submicromolar [Ca2+]c and as an activator of Ca2+ uptake at high [Ca2+]c, and we have used the ability of aequorin to explore different [Ca2+] ranges to study the mechanism of these effects in more detail. In addition, our data reveal several novel surprising details on the mechanism of regulation of MCU.
By studying the mitochondrial Ca2+ uptake induced by a full range of [Ca2+]c, we find that MICU1 knockdown largely activated mitochondrial Ca2+ uptake induced by a low, between 100 nM and approximately 2 μM, [Ca2+]c. In contrast, for a higher [Ca2+]c the rate of mitochondrial Ca2+ uptake was reduced in the MICU1-silenced cells. The results of the present study show that even a [Ca2+] as low as 100 nM triggers a rapid increase in [Ca2+]M in MICU1-silenced cells, whereas it does not produce any effect in the controls. As the [Ca2+]c is increased the rate of mitochondrial Ca2+ uptake rapidly increases in the controls, with a [Ca2+]c greater than 2 μM increasing the [Ca2+]M in the controls to a value higher than the MICU1-silenced cells. However, for a [Ca2+]c between 2 and 5 μM the initial rate of mitochondrial Ca2+ uptake is much faster and starts earlier in the MICU1-silenced cells than in the controls. Therefore the results of the present study suggest that the removal of the MICU1 block of the MCU channel is a time-consuming step that is required to open the MCU. At a [Ca2+]c above 5 μM the difference in the initial rate of mitochondrial Ca2+ uptake is no longer seen, indicating that the rate of removal of the MICU1 block of MCU is much faster at a high [Ca2+]c.
In the present study we have found another unexpected use-dependent regulation of MCU activity, which is particularly apparent in the MICU1-silenced cells. We find that prolonged opening of the MCU by submicromolar [Ca2+] in these cells triggers an inactivation of the mitochondrial Ca2+ entry mechanism activated by MICU1 knockdown. Opening of MCU for 5 min was required for the full inactivation and 50% inhibition was obtained after about 2 min of MCU activation. In addition, once the inactivation has occurred even a prolonged incubation in Ca2+-free medium for up to 15 min was unable to reactivate it. Therefore, in the absence of MICU1, when the MCU remains open for several minutes with a submicromolar [Ca2+]c, a new block appears that restores the impermeability of the MCU at a low [Ca2+]c. This mechanism may represent a safeguard against mitochondrial Ca2+ overload during prolonged increases in [Ca2+]c. The fact that the inactivation requires such a long time to develop suggests that Ca2+ has to enter the mitochondria and trigger it from the inside. In addition, although the inactivation mainly affects Ca2+ entry at a low [Ca2+]c, a smaller effect can be also observed at higher [Ca2+]c values. Thus prolonged opening of the MCU induces a long-lasting MICU1-independent block of the MCU, particularly at a low [Ca2+]c.
This inactivation mechanism recalls some features of the so-called RaM (rapid mode of Ca2+ uptake into mitochondria) . However, some other the mechanisms’ characteristics are different, namely the similar sensitivity to Ruthenium Red of Ca2+ uptake induced by low and high [Ca2+]c values, the longer times required for inactivation, and the apparent lack of reactivation. Regarding the mechanism of the Ca2+-dependent inhibition, we find it is still present in the absence of ATP and in the presence of several antioxidants. These findings suggest that phosphorylation and ROS are probably not involved in the process. Changes in matrix Ca2+ buffering resulting from Ca2+-dependent alkalinization  can also be excluded, because similar [Ca2+]M increases in the control cells did not induce any inhibition. The actual mechanism is therefore still unclear.
In conclusion, opening of the MCU requires removal of the MICU1 block in a time-consuming step and the rate of this step is proportional to the [Ca2+]c. This means that the relative amount of MICU1 expression in a particular tissue may control how mitochondria in different cells respond to fast [Ca2+]c oscillations of low amplitude. Thus a reduced expression of MICU1 would facilitate a more efficient translation to mitochondria of fast and small [Ca2+]c oscillations. In addition, in the absence of MICU1, prolonged activation of the MCU at low [Ca2+]c leads to an inactivation of the channel response to the same range of low [Ca2+]c. This use-dependent inactivation of the MCU may help mitochondria to avoid Ca2+ overload during prolonged increases in [Ca2+]c.
Sergio de la Fuente and Jessica Matesanz-Isabel were responsible for most of the experimental work. Rosalba Fonteriz and Mayte Montero provided additional expertise and laboratory help with most of the experiments. Javier Alvarez directed all of the studies and was primarily responsible for final preparation of the paper before submission, with the assistance of the other authors.
We thank Pilar Alvarez for excellent technical assistance.
This work was supported by the spanish Ministerio de Ciencia e Innovación [grant number BFU2011-25763], the Junta de Castilla y León [grant number VA029A12-1] and the Spanish Government via FPI (Formación de Personal Investigador) fellowships (to S.D.L.F. and J.M.-I.).