Dynamics of matrix-free Ca²⁺ in cardiac mitochondria: two components of Ca²⁺ uptake and role of phosphate buffering

Abstract

Mitochondrial Ca²⁺ uptake is thought to provide an important signal to increase energy production to meet demand but, in excess, can also trigger cell death. The mechanisms defining the relationship between total Ca²⁺ uptake, changes in mitochondrial matrix free Ca²⁺, and the activation of the mitochondrial permeability transition pore (PTP) are not well understood. We quantitatively measure changes in [Ca²⁺]_out and [Ca²⁺]_mito during Ca²⁺ uptake in isolated cardiac mitochondria and identify two components of Ca²⁺ influx. [Ca²⁺]_mito recordings revealed that the first, MCU_mode1, required at least 1 µM Ru360 to be completely inhibited, and responded to small Ca²⁺ additions in the range of 0.1 to 2 µM with rapid and large changes in [Ca²⁺]_mito. The second component, MCU_mode2, was blocked by 100 nM Ru360 and was responsible for the bulk of total Ca²⁺ uptake for large Ca²⁺ additions in the range of 2 to 10 µM; however, it had little effect on steady-state [Ca²⁺]_mito. MCU_mode1 mediates changes in [Ca²⁺]_mito of 10s of μM, even in the presence of 100 nM Ru360, indicating that there is a finite degree of Ca²⁺ buffering in the matrix associated with this pathway. In contrast, the much higher Ca²⁺ loads evoked by MCU_mode2 activate a secondary dynamic Ca²⁺ buffering system consistent with calcium-phosphate complex formation. Increasing P_i potentiated [Ca²⁺]_mito increases via MCU_mode1 but suppressed [Ca²⁺]_mito changes via MCU_mode2. The results suggest that the role of MCU_mode1 might be to modulate oxidative phosphorylation in response to intracellular Ca²⁺ signaling, whereas MCU_mode2 and the dynamic high-capacity Ca²⁺ buffering system constitute a Ca²⁺ sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca²⁺]_mito itself but rather a downstream byproduct of total mitochondrial Ca²⁺ loading.

INTRODUCTION

Ca²⁺ is the central signaling molecule for cardiac excitation-contraction coupling, and its uptake by mitochondria plays a fundamental role in the regulation of ATP production (McCormack et al., 1990; Demaurex and Distelhorst, 2003; Maack and O’Rourke, 2007; Szabadkai and Duchen, 2008). In addition, the extent of mitochondrial Ca²⁺ uptake during metabolic stress determines whether the myocyte lives or dies, owing to the activation of the permeability transition pore (PTP; Crompton, 1999). Although PTP activation is unequivocally linked to total mitochondrial Ca²⁺ load, it is presently unclear how much Ca²⁺ is free or bound in the mitochondrial matrix during PTP activation, as well as how much Ca²⁺ is required to maintain energy balance.

Mitochondrial matrix free Ca²⁺ ([Ca²⁺]_mito) is determined by the balance between Ca²⁺ uptake, extrusion, and buffering. Ca²⁺ uptake is driven by the electrochemical Ca²⁺ gradient and is mediated by a mitochondrial Ca²⁺-selective uniporter (MCU; Gunter and Pfeiffer, 1990), a Ruthenium red (or its potent subcomponent, Ru360)–sensitive ion channel, whose selectivity is determined by nanomolar affinity Ca²⁺ binding to the pore, with an open probability that is voltage dependent (Kirichok et al., 2004). In single-channel mitoplast patch recordings of cardiac mitochondria, two different mitochondrial Ca²⁺ channels, mCU1 and mCU2, with different gating properties and Ru360 sensitivity, were observed (Michels et al., 2009). Two independent groups have recently reported that the molecular identity of the MCU pore is the protein product of the gene CCDC109A (Baughman et al., 2011; De Stefani et al., 2011). Knockdown of this gene suppresses the major component of mitochondrial Ca²⁺ uptake in cultured cells and in the liver (Baughman et al., 2011) and reconstitution of the protein forms Ca²⁺ channels in lipid bilayers (De Stefani et al., 2011). Nevertheless, in addition to the major component of MCU, other potential Ca²⁺ uptake pathways have been noted in mitochondria, including the rapid mode (RaM) of Ca²⁺ uptake (Sparagna et al., 1995; Buntinas et al., 2001) and the mitochondrial ryanodine receptor (mRyR; Beutner et al., 2001; Ryu et al., 2010). The relative importance of each Ca²⁺ uptake pathway, with respect to function, has not yet been determined.

Ca²⁺ extrusion in cardiac mitochondria is determined primarily by the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE; Crompton et al., 1977). Its molecular identity is thought to be an ancestral member of the Na⁺/Ca²⁺ exchanger family, NCLX, a transporter capable of exchanging Ca²⁺ for either Na⁺ or Li⁺ (Palty et al., 2010). The mNCE plays an important role in modulating the steady-state balance between extra- and intramitochondrial Ca²⁺ in isolated mitochondria (Wei et al., 2011), and disruption of this equilibrium in conditions of cellular Na⁺ overload associated with cardiac disease can impact pyridine nucleotide redox and ROS balance in cardiac cells (Maack et al., 2006; Liu and O’Rourke, 2008; Kohlhaas et al., 2010). A minor component of mitochondrial Ca²⁺ efflux is also contributed by the mitochondrial PTP (Wei et al., 2011), which has been proposed to serve as a Ca²⁺ relief valve for mitochondria (Gunter and Pfeiffer, 1990). Disruption of this mechanism can have diverse functional consequences in vivo (Elrod et al., 2010).

The least well understood, but perhaps most important, determinant of [Ca²⁺]_mito is the Ca²⁺ buffering system of the mitochondrial matrix, consisting of both fixed and dynamic buffering mechanisms. The rapid formation of complexes between Ca²⁺ and phosphate (P_i) in mitochondria, described almost 50 yr ago, has been shown to be of major importance in determining how much Ca²⁺ can be accumulated into mitochondria, as well as how much [Ca²⁺]_mito changes in response to total Ca²⁺ taken up (Nicholls and Chalmers, 2004; Chinopoulos and Adam-Vizi, 2010). In fact, in the presence of P_i, but not acetate, as the accompanying permeant anion, mitochondria are capable of sequestering enormous amounts of total Ca²⁺ (>1 M), while maintaining [Ca²⁺]_mito in the low micromolar range, as determined in isolated mitochondria loaded with matrix free Ca²⁺ indicators (Davis et al., 1987; Lukács and Kapus, 1987; Gunter et al., 1988; Lukács et al., 1988; Saavedra-Molina et al., 1990; Walajtys-Rhode et al., 1992; Chalmers and Nicholls, 2003). Interestingly, this range of [Ca²⁺]_mito, <5 µM, appears to be the range over which key Ca²⁺-sensitive mitochondrial enzymes are regulated (Denton et al., 1980; McCormack et al., 1990; Walajtys-Rhode et al., 1992).

In cardiac mitochondria, although many studies have investigated rates of Ca²⁺ uptake, efflux, and PTP activation in isolated mitochondria and intact cells using various methods, the dynamics and quantitative determinants of changes in [Ca²⁺]_mito are incompletely understood. Here, we use matrix-loaded fura-FF, a ratiometric Ca²⁺ indicator with an intermediate Ca²⁺-binding affinity, to quantitatively examine changes in [Ca²⁺]_mito while simultaneously monitoring extramitochondrial Ca²⁺ and ΔΨ_m in isolated guinea pig heart mitochondria. The results indicate that two components of mitochondrial Ca²⁺ uptake are present: one which mediates a large, rapid change in [Ca²⁺]_mito in response to small additions of extramitochondrial Ca²⁺ (MCU_mode1), and another which mediates the stereotypical slow, lower affinity, Ca²⁺ uptake pathway (MCU_mode2) which is capable of taking up large amounts of Ca²⁺ but leads to relatively small changes in [Ca²⁺]_mito. Although MCU_mode2 is completely blocked by 100 nM Ru360, MCU_mode1 has a lower inhibitor affinity, requiring 1 µM Ru360 for complete inhibition. The differential responses of [Ca²⁺]_mito to Ca²⁺ entry via the two different pathways can be explained by a two component buffer system comprised of both static Ca²⁺ buffers and dynamic Ca²⁺ buffering by P_i, which enters in parallel with large amplitude Ca²⁺ influx. The findings have important implications with respect to the physiological regulation of oxidative phosphorylation by Ca²⁺, as well as for the activation of the PTP, which, remarkably, is shown to be uncorrelated with [Ca²⁺]_mito.

MATERIALS AND METHODS

Mitochondria were isolated from adult guinea pig hearts on ice using a homogenization method previously described (Aon et al., 2010). In brief, the heart tissue was homogenized in 75 mM sucrose, 225 mM mannitol, 1 mM HEPES, and 1 mM EGTA, pH 7.4, with 0.1 mg/ml bacterial proteinase type XXIV (Sigma-Aldrich). The homogenate was centrifuged at 480 g for 5 min at 4°C, and the supernatant was centrifuged at 7,700 g for 10 min. The mitochondrial pellet was washed twice at 7,700 g for 5 min and resuspended in sucrose-based isolation solution with 20 µM EGTA to ∼10–20 mg mitochondrial protein/ml (protein concentration was determined by BCA assay). During the experiments, ∼0.6 mg of isolated mitochondria was suspended in 2 ml potassium-based buffer solution composed of: 137 mM KCl, 20 µM EGTA, 20 mM HEPES, and 5 mM glutamate/malate (G/M), with or without 2 mM KH₂PO₄ at pH 7.15.

Multiple mitochondrial parameters, including mitochondrial inner membrane potential (ΔΨ_m) extra- and intramitochondrial Ca²⁺ concentrations, and mitochondrial light scattering, were simultaneously monitored in a stirred cuvette using a fluorometer (QuantaMaster; Photon Technology International) at room temperature. ΔΨ_m was monitored by the dual-excitation ratiometric method with the fluorescent probe tetramethylrhodamine methyl ester (TMRM; 300 nM) at excitations of 546 and 573 nm and emission at 590 nm (Scaduto and Grotyohann, 1999). Mitochondrial 90° light scattering was monitored at 540 nm excitation. The fluorescent Ca²⁺ indicator Calcium green 5N (CaGreen; Invitrogen) was used to monitor extramitochondrial Ca²⁺ ([Ca²⁺]_out), with excitation and emission wavelengths at 505 and 535 nm. Quantitative measurements of [Ca²⁺]_mito were made with the dual-excitation ratiometric Ca²⁺ indicator fura-FF, loaded into the isolated mitochondria as the fura-FF-AM form (20 µM for 30 min at room temperature; washed two to three times with sucrose-based isolation buffer). The fura-FF signal was calibrated in mitochondria treated with the Ca²⁺ ionophore 4-bromo-A23187 (2 µM; Deber et al., 1985; Nicholls and Chalmers, 2004) in the presence of 5 µg/ml oligomycin and 5 µM FCCP to allow equilibration between intra- and extramitochondrial calcium (Wan et al., 1989; Chalmers and Nicholls, 2003; Andrienko et al., 2009; Fig. 1). The calibration curve was established according to the following equation (Grynkiewicz et al., 1985):

$$\left[C{a}^{2+}\right]=K_d‘\beta \frac{(R-R_{\min })}{(R_{\max }-R)},$$

where R is the ratio of 510-nm emission intensities for excitation at 340 and 380 nm. K_d′ is the apparent Ca-fura-FF dissociation constant, and β is the fluorescence intensity ratio for Ca²⁺-free and Ca²⁺-saturated fura-FF excited at 380 nm. R_max and R_min are R values for Ca²⁺-saturated and Ca²⁺-free fura-FF. The calibration was done for fura-FF–loaded mitochondria for each day’s experiment (example shown in Fig. 1). Ratios of background-uncorrected and background-corrected fura-FF fluorescence signal at 340 and 380 nm excitation were fitted to the Grynkiewicz equation (Fig. 1, B and C). Autofluorescence values, and their changes upon Ca²⁺ addition, were relatively small compared with the fura-FF fluorescence intensities (Fig. 1 A, teal and magenta traces) and [Ca²⁺]_mito calculated from calibration curves with or without subtraction of autofluorescence showed no significant differences (Fig. 1 D). Therefore, autofluorescence corrections were not necessary, and were not made, in the subsequent experiments. The method for calibrating the extramitochondrial calcium signal (CaGreen) was as described previously (Wei et al., 2011).

Figure 1.

Calibration of fura-FF signals. (A) Fluorescence recordings of mitochondria in the presence or absence of fura-FF loading (340:510 nm ex:em and 380:510 nm FuraFF or control, respectively) along with [Ca²⁺]_out (CaGreen: 505:535 nm ex:em). The Ca²⁺ signals were calibrated with multiple additions of 5 µM Ca²⁺ in the presence of the ionophore, 2 µM 4-bromo-A23187, 5 µg/ml oligomycin, and 5 µM FCCP. (B) The excitation ratio for fura-FF was calculated with (red) or without (black) correction for autofluorescence. (C) [Ca²⁺]_mito was determined after fitting the calibration curve to the equation [Ca²⁺] = Kd′β (R − R_min)/(R_max − R). The values obtained were R_max = 3.2, R_min = 0.5, K_d′ = 10.7, β = 2.5 without autofluorescence correction (black); R_max = 3.7, R_min = 0.5, K_d′ = 12.7, β = 2.75 for corrected ratio(red). (D) [Ca²⁺]_mito in intact mitochondria after multiple additions of Ca²⁺ (15.3 µM first addition, 5.7 µM for subsequent additions) show that autofluorescence correction had little effect on the calculated [Ca²⁺]_mito.

In the Results and Figure Legends, Ca²⁺ additions to the cuvette are those calculated using the online version of WEBMAXC (http://maxchelator.stanford.edu/webmaxc/webmaxcE.htm). Taking into account the presence of 20 µM EGTA in the experimental solution, this corresponded to free Ca²⁺ changes in the cuvette of 0.011, 0.2, 0.55, 2, 5.7, and 15.3 µM for additions of 1, 10, 15, 20, 25, and 35 µM Ca²⁺, respectively. Because other low-affinity Ca²⁺ buffers in the solution, such as glutamate and malate, were not included in this calculation, these numbers tend to overestimate the free Ca²⁺ change for larger Ca²⁺ additions, explaining discrepancies in the direct measurements of initial [Ca²⁺]_out using calibrated CaGreen signals. Mitochondrial matrix calcium buffering, i.e., the relationship between the total Ca²⁺ taken up by mitochondria and the change in [Ca²⁺]_mito, was expressed as the ratio of bound/free Ca²⁺ and was examined in the presence of different anions, including P_i (0.1–10 mM), arsenate (As_i; 2 mM), or vanadate (V_i; 2 mM). Cyclosporin A (CsA; 1 µM) was used to shift the threshold for activation of the PTP to a higher Ca²⁺ range to study Ca²⁺ dynamics over a wide range of mitochondrial Ca²⁺ loads. The figures show representative responses for each experiment; however, every experiment was repeated at least three times with a similar result (see Fig. S1 for an example of the range of statistical variability of the response).

Online supplemental material

Fig. S1 shows the mean and standard error of the response of [Ca²⁺]_mito to large Ca²⁺ additions (n = 4 experiments). Fig. S2 shows the lack of a ryanodine effect on mitochondrial Ca²⁺ for a single Ca²⁺ addition. Fig. S3 shows Na⁺/Ca²⁺ exchanger effects on mitochondrial Ca²⁺ with different concentrations of Na⁺ and in the presence of the inhibitor CGP37157. Online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.201210784/DC1.

RESULTS

In Fig. 2 (top), simultaneous recordings of [Ca²⁺]_out (green traces), [Ca²⁺]_mito (black traces), and ΔΨ_m (red traces) are shown superimposed for large (Fig. 2, A–D) or small (Fig. 2, E–H) Ca²⁺ additions to the cuvette (from a starting [Ca²⁺]_out of ∼0.100 µM before the first addition). Immediately evident is the fact that two components of the mitochondrial Ca²⁺ response are present. For the series of larger Ca²⁺ additions in Fig. 2 A, although the first Ca²⁺ addition (15.3 µM) evoked a large increase in [Ca²⁺]_mito to ∼50 µM, subsequent additions (5.7 µM each) caused a transient increase (amplitude 10–20 µM) in [Ca²⁺]_mito, followed by a decline back to the same 50 µM steady-state matrix concentration. Moreover, after the sixth Ca²⁺ addition, this steady-state [Ca²⁺]_mito begins to decrease after each pulse (to ∼20 µM), suggesting an increase in the matrix Ca²⁺ buffer capacity (changes in matrix volume, as assessed by light scattering, could not account for these changes in [Ca²⁺]_mito, compare Fig. 8). ΔΨ_m depolarized by 5–10 mV with each Ca²⁺ addition and recovered back to 180 mV as extramitochondrial Ca²⁺ (and MCU flux, which follows the electrochemical gradient for Ca²⁺) returned to a low level. Comparing the change in matrix free Ca²⁺ (expressed as nmols/mg mitochondrial protein assuming 1 µl/mg mitochondrial volume; Fig. 2 B) to the total amount of Ca²⁺ taken up from the bath (ΣCa²⁺_uptake; Fig. 2 C) allowed us to calculate the ratio of bound/free Ca²⁺ (Fig. 2 D), illustrating how the buffer capacity dramatically increases after the first Ca²⁺ addition. The initial rapid jump in [Ca²⁺]_mito (MCU_mode1), which reached completion well before the end of the major slow uptake phase of the uniporter (MCU_mode2), prompted us to explore the [Ca²⁺]_mito response to a much lower range of Ca²⁺ concentrations.

Figure 2.

[Ca²⁺]_mito (black), [Ca²⁺]_out (green), and ΔΨ_m (red) responses to large or small Ca²⁺ additions in isolated guinea pig heart mitochondria. (A–D) Ca²⁺ additions were as follows: 15.3 µM Ca²⁺ first addition, 5.7 µM for each subsequent addition. (E–H) Ca²⁺ additions were as follows: 0.2 µM Ca²⁺ first addition, 0.011 µM for each subsequent addition. Corresponding calculated values of free Ca²⁺_mito expressed in nmol/mg, total ∑Ca²⁺ taken up, and the bound/free Ca²⁺ ratio are shown for large (A–D) or small (E–H) additions. [Ca²⁺]_mito and [Ca²⁺]_out were monitored with Calcium green and fura-FF simultaneously. Experimental solution was the standard KCl-based buffer with 5 mM NaCl, 5 mM G/M, 1 mM KH₂PO₄, 20 µM EGTA, and 1 µM CsA. 300 nM TMRM was used to monitor ΔΨ_m ratiometrically.

Figure 8.

Effects of P_i, (black), arsenate (As_i, blue), or vanadate (V_i, red) anions on the regulation of mitochondrial Ca²⁺. The buffer solution contained 2 mM P_i, As_i, or V_i in the presence of 1 µM of CsA. (A–E) Large Ca²⁺ additions (first Ca²⁺ addition was 15.3 µM, subsequent additions 5.7 µM each) were made until mitochondrial PTP opening occurred, as indicated by ΔΨ_m collapse (C), swelling (D), and uncontrolled Ca²⁺ efflux (A). (F–J) Smaller Ca²⁺ additions (first Ca²⁺ addition 0.2 µM, subsequent 0.011 µM each) evoked similar [Ca²⁺]_mito responses without significantly disrupting ΔΨ_m or activating PTP.

Fig. 2 E shows that adding 0.2 µM Ca²⁺ first allows one to resolve the kinetics of the initial high-affinity Ca²⁺ uptake. [Ca²⁺]_mito increased over the course of 100 s (as compared with the <10-s increase for the 15.3-µM addition in Fig. 2 A) and responded in a stepwise fashion for two subsequent additions of 0.01 µM each. Six more 0.01 µM Ca²⁺ additions evoked transient increases in [Ca²⁺]_mito but little change in the steady-state [Ca²⁺]_mito until the ninth addition, when the steady state actually began to decline. ΔΨ_m decreased by only 3 mV (188 to 185mV) after the first Ca²⁺ addition but did not change for subsequent additions (Fig. 2 E). Interestingly, despite the 100-fold lower amount of total Ca²⁺ loaded into the mitochondria in the small (Fig. 2 G) versus large (Fig. 2 C) Ca²⁺ addition experiments, the steady-state [Ca²⁺]_mito, in both cases, settled into a range between 15 and 20 µM, demonstrating that matrix Ca²⁺ buffering dynamically responds over a very wide range. This is clearly evident when one compares the changes in matrix free Ca²⁺ (Fig. 2, B and F) with the total Ca²⁺ taken up by the mitochondria (Fig. 2, C and G), expressed as the ratio of bound/free Ca²⁺ (Fig. 2, D and H). This ratio spanned from ∼3 to 30,000 over the range of Ca²⁺ additions examined.

We next examined the properties of MCU_mode1 in greater detail for an initial Ca²⁺ addition of 2 µM. CGP-37157, cyclosporin A, and thapsigargin were also included in the experimental solution to inhibit possible Ca²⁺ fluxes through the mNCE, PTP, and the sarcoplasmic reticular Ca²⁺ ATPase, respectively. Ru360 is a derivative of Ruthenium red that potently inhibits mitochondrial Ca²⁺ uptake with a K_D of ∼1.3 nM (Ying et al., 1991). In the absence of Ru360, [Ca²⁺]_mito (Fig. 3 B, black trace) increased rapidly after the Ca²⁺ addition and then reached a plateau, even though extramitochondrial Ca²⁺ decreased continuously over 50–100 s (Fig. 3 A, black trace). At Ru360 concentrations of 2 and 10 nM (Fig. 3 B, red and blue traces), the rate of rise of MCU_mode1 was slowed but its plateau was unchanged. In contrast, MCU_mode2 was markedly inhibited (the initial rate of decline of [Ca²⁺]_out decreased by almost 95%) at 10 nM Ru360 (Fig. 3 A, blue trace) and was completely blocked by 100 nM Ru360 (Fig. 3 A, magenta trace). MCU_mode1, in contrast, was only partially inhibited by 100 nM Ru360, and 1 µM Ru360 was required for complete inhibition (Fig. 3 B).

Figure 3.

Concentration dependence of Ru360 inhibition of mitochondrial Ca²⁺ uptake. Fura-FF–loaded mitochondria were preincubated with 0 (black), 2 (red), 10 (blue), 100 (magenta), or 1,000 nM (green) Ru360 in the standard KCl-based buffer with 2 mM KH₂PO₄, 5 mM G/M, 5 mM NaCl, 10 µM CGP37157, 1 µM CsA, and 2.5 µM thapsigargin. 2 µM free Ca²⁺ was added to initiate mitochondrial Ca²⁺ uptake and [Ca²⁺]_out (A) and [Ca²⁺]_mito (B) were monitored simultaneously.

The greater sensitivity of MCU_mode2 to inhibition by Ru360 allowed us to investigate the Ca²⁺ dependence of MCU_mode1 with MCU_mode2 largely inhibited. Fig. 4 shows the [Ca²⁺]_mito response to Ca²⁺ additions in the range of 0.2 to 5.7 µM Ca²⁺ in the absence (Fig. 4 A) and presence (Fig. 4 B) of 10 nM Ru360. With MCU_mode2 almost completely inhibited by Ru360, as indicated by the near step-like changes in cuvette Ca²⁺ upon Ca²⁺ addition (Fig. 4 B, bottom), the rate of rise of MCU_mode1 increased as a function of Ca²⁺ and the plateau level of [Ca²⁺]_mito increased to ∼40 µM for additions of 2 and 5.7 µM (Fig. 4 B, top). Remarkably, although much more total Ca²⁺ uptake occurred with both MCU_mode1 and MCU_mode2 active in the absence of Ru360 (Fig. 4 A, bottom), the change in plateau [Ca²⁺]_mito, presumably mediated only by MCU_mode1, was very similar, reaching a plateau of ∼40 µM free Ca²⁺ (Fig. 4 A, top). The rate of rise of MCU_mode1 was ∼10-fold faster in the absence of 10 nM Ru360; however, the Ca²⁺ dependence of the rate was similar. Steady-state ΔΨ_m was maintained at close to 185 mV during these experiments (unpublished data). The results indicate that MCU_mode1 underlies the largest change in [Ca²⁺]_mito after a Ca²⁺ addition to isolated cardiac mitochondria, whereas the large amount of Ca²⁺ taken up via MCU_mode2 is almost entirely buffered.

Figure 4.

Calcium dependence of mitochondrial Ca²⁺ uptake in the absence (A) and presence (B) of 10 nM Ru360. [Ca²⁺]_out and [Ca²⁺]_mito were monitored during mitochondrial Ca²⁺ uptake initiated by Ca²⁺ additions ranging from 0.2 (black), to 0.55 (red), to 2 (blue), to 5.7 µM (green). 10 nM Ru360 largely eliminated MCU_mode2 (see [Ca²⁺]_out in bottom panels) but only partially inhibited MCU_mode1 ([Ca²⁺]_mito response in top panels). Standard KCl-based buffer with 2 mM KH₂PO₄, 5 mM G/M, 5 mM NaCl, 10 µM CGP, 1 µM CsA, and 2.5 µM thapsigargin.

The experiments shown in Fig. 5 were designed to test (1) whether the initial jump in [Ca²⁺]_mito mediated by MCU_mode1 could be reset and repeated if cuvette Ca²⁺ was lowered by EGTA addition, and (2) whether Ca²⁺ efflux from the compartment reported by fura-FF was mediated by the mNCE. Isolated mitochondria were first subjected to a single addition of Ca²⁺ (2 µM) to initiate mitochondrial Ca²⁺ uptake. EGTA was then added to decrease [Ca²⁺]_out back to the nM range (∼100 nM) and a second Ca²⁺ addition was made (4 µM). In the absence of extramitochondrial Na⁺, [Ca²⁺]_out declined with a time course similar to the experiments described in the previous paragraphs and [Ca²⁺]_mito again showed a rapid increase via MCU_mode1 to reach a plateau (Fig. 5 A). EGTA addition resulted in a rapid decrease in [Ca²⁺]_out but [Ca²⁺]_mito remained elevated (Fig. 5 A). A second Ca²⁺ addition effected a transient in [Ca²⁺]_out similar to the first addition; however, instead of a stepwise increase in [Ca²⁺]_mito the second pulse evoked a spike and decay response of [Ca²⁺]_mito. Apparently, when [Ca²⁺]_mito is already elevated, additional Ca²⁺ entering via MCU is buffered after a transient response. In contrast, in the presence of Na⁺, both [Ca²⁺]_out and [Ca²⁺]_mito decline after EGTA addition (Fig. 5 B). A second Ca²⁺ addition in this case evokes a [Ca²⁺]_mito response similar to the first pulse, i.e., a rapid rise and plateau. Collectively, these results show that the rapid rise in [Ca²⁺]_mito via MCU_mode1 is best observed when [Ca²⁺]_out is increased from the nanomolar to the micromolar range, and when the total mitochondrial Ca²⁺ load is low. Moreover, the Ca²⁺ compartment filled via MCU_mode1 is emptied via mNCE, confirming that it is either the same compartment that is filled by MCU_mode2, or it has Ca²⁺ efflux pathways identical to the latter.

Figure 5.

Resetting of [Ca²⁺]_mito (black) for two Ca²⁺ additions. 2 µM Ca²⁺ was first added to initiate mitochondrial Ca²⁺ uptake. 25 µM EGTA was added to decrease the extramitochondrial calcium concentration ([Ca²⁺]_out, green) to the submicromolar range, and another 4 µM Ca²⁺ was added to evoke a second [Ca²⁺]_mito response. A decrease in [Ca²⁺]_mito upon lowering [Ca²⁺]_out occurred only in the presence of Na⁺. Conditions: (A) zero NaCl; (B) 15 mM NaCl; (C) 15 mM NaCl plus 100 nM Ru360; (D) 15 mM NaCl plus 2 mM MgATP.

The same two-pulse protocol evoked two increases in [Ca²⁺]_mito that plateaued after ∼100 s with MCU_mode2 completely blocked with 100 nM Ru360 (Fig. 5 C). This residual uptake was likely to be mediated by MCU_mode1, whose kinetics were slowed by 100 nM Ru360, in accordance with the data shown in Fig. 3 B. Finally, the same protocol was applied in a more physiological condition in the presence of 2 mM MgATP (free Mg²⁺ was calculated to be 0.467 mM, which is close to that present in the cytoplasm). In the presence of MgATP, the rates of mitochondrial Ca²⁺ uptake via MCU_mode1 and MCU_mode2 were both decreased (Fig. 5 D), but the Na⁺-dependent Ca²⁺ efflux rate and qualitative responses to the sequential pulses were similar to those in the absence of MgATP (compare Fig. 5 B).

The slowing of the Ca²⁺ uptake rate with MgATP could be attributed to the increased Mg²⁺ present in the solution (Fig. 6). The physiological Mg²⁺ concentration in cytosol is ∼0.1–0.8 mM (Szanda et al., 2009) and Mg²⁺ is known to have inhibitory effects on mitochondrial Ca²⁺ uptake (Crompton et al., 1975; Bragadin et al., 1979). The effects of Mg²⁺ (from 0.1 to 1.0 mM) on mitochondrial Ca²⁺ uptake and [Ca²⁺]_mito were compared for both small and large Ca²⁺ additions. The mitochondrial Ca²⁺ uptake rate and [Ca²⁺]_mito were minimally affected for additions of 15 µM Ca²⁺ (Fig. 6, A–C); however, with smaller Ca²⁺ additions (first Ca²⁺ addition, 0.2 µM; subsequent, 0.011 µM each), Mg²⁺ slowed mitochondrial Ca²⁺ uptake (Fig. 6, D and E). Decreased uptake was indicated by higher [Ca²⁺]_out and lower [Ca²⁺]_mito in the presence of high Mg²⁺. The finding that Mg²⁺ inhibition can be overcome at high Ca²⁺ suggests that Mg²⁺ is competitive with respect to Ca²⁺ flux through the MCU.

Figure 6.

Effect of Mg²⁺ on the regulation of mitochondrial Ca²⁺. (A–C) Large Ca²⁺ additions (Ca²⁺ additions were 15.3 µM each) in the presence of zero (black), 0.1 (blue), or 1 mM (red) MgCl_2. (D–F) Small Ca²⁺ additions (first Ca²⁺ addition 0.2 µM, subsequent 0.011 µM each) with 0 (black), 0.1 (green), 0.5 (blue), and 1 mM (red) MgCl₂.

The rapid buffering of [Ca²⁺]_mito during large increases in total mitochondrial Ca²⁺ evident in the preceding experiments prompted us to investigate the effects of changing the anion concentration on matrix Ca²⁺ dynamics. Because the formation of calcium phosphate complexes is thought to be the major mitochondrial Ca²⁺ buffering reaction (Nicholls and Chalmers, 2004), we examined the effects of varying P_i over the range of 0.1 to 10 mM for both large and small Ca²⁺ additions. In 0.1 mM P_i, the first large Ca²⁺ addition (15.3 µM) increased [Ca²⁺]_mito immediately to ∼50 µM (Fig. 7 A; black trace), and subsequent Ca²⁺ additions (5.7 µM each) induced transients in [Ca²⁺]_mito with increasing amplitudes of >100 µM. Nevertheless, [Ca²⁺]_mito returned to a matrix setpoint of 50 µM after each transient. Ca²⁺ additions in the presence of 0.1 mM P_i evoked deeper and more prolonged depolarizations in ΔΨ_m (Fig. 7 A; red trace) than those observed for 1 mM (Fig. 7 B) or 10 mM (Fig. 7 C) P_i, increasing in amplitude from 20 to 40 mV with each subsequent addition. The overall rate of mitochondrial Ca²⁺ uptake was accelerated in 1 mM (Fig. 7 B) and 10 mM (Fig. 7 C) P_i, as compared with the rate in 0.1 mM P_i, and the rate of return of [Ca²⁺]_mito to the matrix Ca²⁺ setpoint was accelerated. Moreover, the [Ca²⁺]_mito setpoint achieved was lowered to 30 µM in 1 mM Pi and to 11 µM in 10 mM Pi by the end of the protocol. ΔΨ_m was well maintained in both 1 and 10 mM Pi (Fig. 7, B and C). Thus, the Ca²⁺ buffering action of P_i facilitates the regulation of [Ca²⁺]_mito down to low levels in the face of very high mitochondrial Ca²⁺ loads and preserves the energy state of the mitochondria.

Figure 7.

Inorganic phosphate (P_i) dependence of mitochondrial Ca²⁺ uptake for large or small Ca²⁺ additions. Isolated mitochondria were incubated in 0.1 (A), 1 (B), or 10 (C) mM P_i with 5 mM NaCl, 5 mM G/M, and 1 µM CsA present. Ca²⁺ additions: First Ca²⁺ addition was 15.3 µM, subsequent additions 5.7 µM each (A–C); red line represents mitochondrial membrane potential ΔΨ (D–E). First Ca²⁺ addition 0.2 µM, subsequent 0.011 µM each. The black, red, blue lines represent 0.1, 1.0, and 10.0 mM P_i, respectively. (E) 10 nM Ru360-treated mitochondria.

For small Ca²⁺ additions (0.2 µM first, with subsequent additions of 0.011 µM each), P_i had a different effect on [Ca²⁺]_mito (Fig. 7, D and E). In this case, increasing P_i from 0.1 to 1 to 10 mM markedly enhanced the rise in [Ca²⁺]_mito associated with each Ca²⁺ addition and [Ca²⁺]_mito was not regulated down to the low levels observed after larger Ca²⁺ additions. The enhanced Ca²⁺ uptake at high P_i was indirectly confirmed by the fact that [Ca²⁺]_out was also observed to be lower in 10 mM P_i (Fig. 7 D, bottom). With MCU_mode2 inhibited with 10 nM Ru360, the potentiating effect of P_i on mitochondrial Ca²⁺ uptake for the high-affinity uptake pathway was still present (Fig. 7 E). These results illustrate the complexity of the mitochondrial matrix Ca²⁺ buffering system, which depends on the amplitudes and rates of entry of both Ca²⁺ and P_i.

Finally, the effect of varying the chemical nature of the buffering anion was explored by comparing the effects of P_i with those of the P_i analogues, vanadate (V_i), or arsenate (As_i; Fig. 8). For large Ca²⁺ additions (Fig. 8, A–E), the most obvious effect of substituting P_i was that the total Ca²⁺ load at which PTP was triggered was markedly lower with V_i or As_i, as indicated by loss of the ability to retain matrix Ca²⁺ (Fig. 8, A and B), ΔΨ_m depolarization (Fig. 8 C), and rapid mitochondrial swelling (Fig. 8 D). Interestingly, this PTP sensitization could not be directly attributed to differences in [Ca²⁺]_mito; V_i substitution lowered [Ca²⁺]_mito and substantially decreased the rate of mitochondrial Ca²⁺ uptake, whereas As_i potentiated [Ca²⁺]_mito without having a significant effect on the uptake rate. Matrix Ca²⁺ buffering, as indicated by the bound/free Ca²⁺ ratio (Fig. 8 E), was more effective for V_i and less effective for As_i for the first few Ca²⁺ additions, as compared with P_i, although just before the activation of PTP in V_i and As_i, bound/free Ca²⁺ was similar for all three anions. Striking differences in the extent of ΔΨ_m depolarization during Ca²⁺ uptake were evident when P_i was substituted with As_i or V_i (Fig. 8 C). V_i substitution had the largest effect, causing a ΔΨ_m depolarization of >30 mV after the first Ca²⁺ addition, whereas the depolarization was only 2 mV in P_i. With smaller Ca²⁺ additions, there was little effect of anion substitution on the ΔΨ_m (Fig. 8 H) or light scattering responses (Fig. 8 I), but just as for the larger Ca²⁺ pulses, [Ca²⁺]_mito was lower in V_i.

DISCUSSION

In the present work, we used calibrated Ca²⁺-sensitive fluorescent indicators to quantitatively measure changes in [Ca²⁺]_out and [Ca²⁺]_mito (as well as ΔΨ_m and light scattering in some experiments) during mitochondrial Ca²⁺ uptake in isolated mitochondria. This method allowed us to identify two components of Ca²⁺ influx that have: (1) very different effects on matrix free [Ca²⁺], (2) different sensitivities to inhibition by Ru360, (3) different affinities for Ca²⁺, and (4) different responses to a change in P_i. MCU_mode1 required at least 1 µM Ru360 to be completely inhibited, and responded to small Ca²⁺ additions in the range of 0.1 to 2 µM with rapid and large changes in [Ca²⁺]_mito. MCU_mode2 was blocked by <100 nM Ru360, was responsible for the bulk of total Ca²⁺ uptake for large Ca²⁺ additions in the range of 2 to 15 µM, and took >100 s to reach steady state. The finding that the MCU_mode1 could mediate changes in [Ca²⁺]_mito of 10 s of μM, even in the presence of 100 nM Ru360, indicates that at low total Ca²⁺ loads there is a finite degree of Ca²⁺ buffering in the matrix; however, at the much higher Ca²⁺ loads supported by MCU_mode2, a secondary dynamic Ca²⁺ buffering system involving P_i is engaged. The results suggest that the role of MCU_mode1 might be to modulate oxidative phosphorylation in response to intracellular Ca²⁺ signaling, whereas MCU_mode2, and the dynamic high-capacity Ca²⁺ buffering system constitutes a Ca²⁺ sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca²⁺]_mito itself but rather a downstream byproduct of matrix Ca²⁺ loading.

Several earlier studies have used AM loading of Ca²⁺ indicators in isolated mitochondria to qualitatively assess matrix Ca²⁺ responses (Davis et al., 1987; Lukács and Kapus, 1987; Gunter et al., 1988; Lukács et al., 1988). A key question that one must first answer before interpretations about matrix Ca²⁺ can be made is whether the dye is localized exclusively in the matrix, or if a certain fraction of the signal is coming from a nonmatrix compartment. Our evidence conclusively demonstrates that the fura-FF is reporting matrix Ca²⁺. Both the fast and slow components of the [Ca²⁺]_mito signal can be completely blocked by Ru360, albeit with different sensitivities. In the case of MCU_mode1 in particular, the kinetics were slowed by Ru360 in a concentration-dependent manner (Fig. 3 B). The initial rate of rise of MCU_mode1 was approximately one order of magnitude slower in 100 nM Ru360 for a range of Ca²⁺ additions (Fig. 4). In addition, the depolarization of ΔΨ_m upon Ca²⁺ addition tracks the rapid component of the [Ca²⁺]_mito response. This dissipation of the protonmotive force can occur only when Ca²⁺ current is flowing across the inner membrane. Fourth, when [Ca²⁺]_out is lowered by EGTA addition to the cuvette, [Ca²⁺]_mito remains high (Fig. 5 A) in the absence of Na⁺ because the mNCE is disabled, but it declines when mNCE is functional (Fig. 5 B). Thus, all of the evidence is consistent with matrix localization of the fura-FF.

Rapid effects of Ca²⁺ on mitochondrial function have been known since 1965 when Chance (1965) reported that Ca²⁺ was able to induce a “state 4 to state 3” respiratory transition in a manner similar to, but slightly faster than, ADP. Ca²⁺ addition (200 µM) by means of a rapid mixing device caused a cytochrome b oxidation response with a halftime of 70 ms. Territo et al. (2001) showed that the initial response time of mitochondrial Ca²⁺-stimulated respiration and light scattering changes is <300 ms for Ca²⁺ additions in the range of ∼60 to 2,000 nM and also reported that a 535-nM Ca²⁺ addition increased [Ca²⁺]_mito by ∼700 nM (as measured using Rhod-2). Gunter and Pfeiffer (1990) and Sparagna et al. (1995) proposed that two pathways for mitochondrial Ca²⁺ uptake exist, a RaM which responds to fast transients of Ca²⁺ in the physiological range (100–500 nM) and the classical MCU which has a low affinity and slow kinetics. RaM-mediated uptake was complete in only 0.3 s and was inactivated when the prepulse steady-state Ca²⁺ exceeded 150 nM. Its amplitude could be fully reset after 1 min in <100 nM Ca²⁺ in cardiac mitochondria (Buntinas et al., 2001). Interestingly, RaM was found to be less sensitive to Ruthenium red than MCU (Sparagna et al., 1995; Buntinas et al., 2001). Hence, several aspects of MCU_mode1 in the present study are similar to the properties of RaM, for example, a lower sensitivity to Ru360 and a higher Ca²⁺ affinity than MCU_mode2. However, in the present study, MCU_mode1 kinetics were slower to reach steady state. For example, in Fig. 2 E, there is a very rapid component of initial uptake for a Ca²⁺ addition of 200 nM, but [Ca²⁺]_mito was still increasing 100 s after the addition and did not appear to be inactivated in the way that RaM would be when subsequent Ca²⁺ additions were made (Fig. 2 F).

Whether MCU_mode1 and MCU_mode2 are two modes of the same Ca²⁺ channel or are separate proteins remains to be determined. The recent discovery that knockdown of the product of the gene CCDC109A (renamed MCU) suppresses MCU_mode2 (Baughman et al., 2011; De Stefani et al., 2011) might allow us to answer this question in future studies. Nevertheless, two Ca²⁺ channels (mCa1 and mCa2) with different gating and conductance properties have been observed recently in mitoplast patch clamp studies of cardiac mitochondria (Michels et al., 2009). MCa1 was inhibited by 200 nM Ru360, whereas mCa2, which had a lower conductance, required 10 µM Ru360 to be suppressed, providing some evidence to suggest that two independent Ca²⁺ channels with different Ru360 sensitivities exist in the inner membrane. The skeletal muscle isoform of the ryanodine receptor has also been proposed as an alternative mitochondrial Ca²⁺ uptake protein. We did not observe any effect of 100 µM ryanodine on Ca²⁺ uptake kinetics in our experiments (Fig. S2).

The remarkable finding of the present study was that small amounts of Ca²⁺ uptake lead to large changes in [Ca²⁺]_mito, whereas large Ca²⁺ additions give only a transient increase in [Ca²⁺]_mito with no change or an even a lower steady-state Ca²⁺. Two possible explanations could account for the paradoxical behavior of [Ca²⁺]_mito; either the Ca²⁺ efflux rate from the mitochondria increases to match the Ca²⁺ entering for large Ca²⁺ loads, or Ca²⁺ buffering in the matrix is markedly increased in parallel with Ca²⁺ entry. The former explanation can be ruled out because we simultaneously measured [Ca²⁺]_out, which confirmed that the Ca²⁺ taken up was retained inside the mitochondria. Under zero Na⁺ conditions, or in the presence of CGP-37157, an inhibitor of mNCE, [Ca²⁺]_mito, was minimally affected (Fig. S3), although there was an effect of Na⁺ on the extramitochondrial Ca²⁺ steady-state concentration, as we have previously described (Wei et al., 2011). The only remaining explanation is that above a certain threshold of Ca²⁺ uptake, the Ca²⁺ buffer capacity increases rapidly to counteract MCU_mode2-mediated uptake. This behavior is similar to that reported by Chalmers and Nicholls (2003) for rat liver and brain mitochondria. They noted that, over a wide range of mitochondrial Ca²⁺ loads (from 10 to 500 nmol/mg), mitochondria were capable of maintaining [Ca²⁺]_mito within a narrow range between 1 and 5 µM, whereas <10 nmol/mg [Ca²⁺]_mito was more linearly related to the magnitude of the uptake. Because the dynamic buffering occurred in the presence of P_i, but not when acetate was the anion, they interpreted the buffering mechanism to be related to the formation of calcium-phosphate complexes, in accordance with other studies (David, 1999; Nicholls, 2005; Warashina, 2006). Our data are consistent with this interpretation but differ in the quantitative details. For small Ca²⁺ additions, [Ca²⁺]_mito increased in a stepwise manner from a baseline of ∼2.5 µM up to ∼25 µM before the secondary buffering took effect and a plateau was reached (Fig. 2 E). This corresponded to an increase in calcium-phosphate buffering occurring after a total Ca²⁺ load of 0.5 nmol/mg (Fig. 2 G). This threshold is likely to vary depending on how the Ca²⁺ addition is made. For example, the larger additions evoked only a single rapid jump in [Ca²⁺]_mito to ∼60 µM (Fig. 2 A) at a total load of 25 nmol/mg, followed by more and more efficient buffering up to 100 nmol/mg (Fig. 2 C). The bound/free Ca²⁺ ratio increased continuously to a level >30,000 as ΣCa²⁺_uptake increased. Increasing P_i over a range from 0.1 to 10 mM did, in fact, result in a lower steady-state [Ca²⁺]_mito for large Ca²⁺ additions (Fig. 7, A–C). In addition, at 0.1 mM P_i, the rate of Ca²⁺ uptake through MCU_mode2 was markedly depressed, ΔΨ_m depolarization was deeper and slower to recover, and [Ca²⁺]_mito rose dramatically for the later Ca²⁺ additions. The slower kinetics of MCU_mode2 in 0.1 mM P_i could be related to a decreased rate of calcium-phosphate formation, which would limit the influx rate by decreasing the electrochemical driving force for Ca²⁺ entry, or it could be a result of the Ca²⁺ influx rate being limited by the slow kinetics of the P_i transport process because anion flux would be required to maintain electroneutrality during movement of the divalent cation. In contrast to the P_i effect on large Ca²⁺ additions, P_i enhanced the rise in [Ca²⁺]_mito with each small Ca²⁺ addition. This was the result of enhanced total Ca²⁺ uptake rather than a change in matrix buffering, as indicated by lower [Ca²⁺]_out at high P_i (Fig. 7, D and E). Facilitation of MCU_mode1 by P_i was even present when MCU_mode2 was blocked by Ru360.

Substitution of P_i with As_i or V_i had striking effects on [Ca²⁺]_mito and the threshold for PTP activation. Interestingly, As_i and V_i had opposite effects on [Ca²⁺]_mito for multiple pulses but they both sensitized the mitochondria to PTP activation. Although As_i appeared to substitute reasonably well for P_i for the first six Ca²⁺ additions with respect to the overall kinetics of MCU_mode2, V_i substitution slowed Ca²⁺ uptake and inhibited the rise in [Ca²⁺]_mito. Even though the total Ca²⁺ uptake was lower in V_i, the PTP activation threshold was the lowest of the three anions. In accord with the conclusions of Chalmers and Nicholls (2003), PTP activation was, in all cases, independent of [Ca²⁺]_mito. Therefore, an alternative hypothesis is required to explain why the PTP is activated at a reproducible total mitochondrial Ca²⁺ load. One possible mechanism could be related to the formation of Ca²⁺-phosphate species or Ca²⁺-polyphosphate complexes (Pavlov et al., 2005; Abramov et al., 2007). If this is the case, both As_i and V_i appear to be adequate (or better) substitutes for P_i in terms of triggering PTP. Recently, Basso et al. (2008) reported that the desensitizing effect of CsA on PTP activation by Ca²⁺ is absent when As_i or V_i are substituted for P_i. Our results confirm this finding and argue against the potential explanation that differences in Ca²⁺ buffering and [Ca²⁺]_mito could be behind the As_i and V_i effects.

Based on the experimental observations, we propose that mechanisms of mitochondrial Ca²⁺ uptake and matrix Ca²⁺ buffering involve two stages (summarized in Fig. 9): (1) Ca²⁺ uptake mediated by MCU_mode1 for extramitochondrial Ca²⁺ changes in the physiological range of cytoplasmic Ca²⁺ that lead to a rise in [Ca²⁺]_mito as the fixed Ca²⁺ buffers become saturated, and (2) Ca²⁺ uptake mediated by MCU_mode2 that is accompanied by rapid buffering via calcium-phosphate complex formation with little change in [Ca²⁺]_mito. Importantly, the MCU_mode1 pathway would be accelerated by nearby Ca²⁺ release into a local extramitochondrial microdomain (Hajnóczky et al., 2000), explaining observations of rapidly rising mitochondrial Ca²⁺ transients during cardiac excitation-contraction coupling (Maack et al., 2006). The magnitude of changes in [Ca²⁺]_mito brought about by MCU_mode1 would fulfill the role of a signal to increase oxidative phosphorylation and ATP production (Bell et al., 2006), as they span the range of activation of the Ca²⁺ dependent dehydrogenases, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase (McCormack et al., 1990), and/or Ca²⁺-dependent allosteric regulatory sites on the respiratory chain complexes (Territo et al., 2000). In contrast, MCU_mode2, along with the rapid dynamic Ca²⁺ buffering mechanism, fulfills the role of a protective Ca²⁺ sink for the cell, enabling the cell to survive in the presence of rather large total Ca²⁺ loads, for example, those that might be encountered in the heart during ischemia-reperfusion, tachycardia, or maximal sympathetic stimulation. In this scenario, the major mitochondrial Ca²⁺ efflux pathway, mNCE, would serve as a modulator of the balance between cytoplasmic Ca²⁺ and matrix Ca²⁺ because its capacity to counteract large MCU fluxes is limited (and would tend to cause cytoplasmic Ca²⁺ overload if its Vmax was fast enough to match MCU-mediated influx). PTP activation can be clearly dissociated from [Ca²⁺]_mito overload by itself but does correlate well with a reproducible threshold of total mitochondrial Ca²⁺ loading. This suggests that a downstream byproduct of Ca²⁺, perhaps a Ca²⁺-phosphate species itself, triggers permeabilization of the inner membrane. How cyclophilin d modulates this trigger remains a mystery because it seems that substitution of P_i with As_i or V_i bypasses the inhibition by cyclosporin A.

Figure 9.

Summary of the mechanisms governing mitochondrial Ca²⁺ dynamics. Mitochondrial Ca²⁺ uptake occurs through MCU_mode1 with a high Ca²⁺ affinity, but low Ru360 sensitivity, whereas the bulk of mitochondrial Ca²⁺ loading occurs through the low Ca²⁺ affinity, high Ru360-sensitive MCU_mode2. Ca²⁺ extrusion occurs through the mNCE. Rapid Ca²⁺ entry via MCU_mode1 saturates fixed mitochondrial Ca²⁺ buffers (B_m) to produce a large change in [Ca²⁺]_mito, whereas larger amounts of Ca²⁺ entry, along with P_i uptake, result in reversible precipitation of calcium-phosphate (CaP_pt) with little effect on steady-state [Ca²⁺]_mito. P_i is transported into mitochondria through the phosphate carrier (PiC). Mitochondrial matrix contents are released when the mitochondrial PTP opens. PTP opening correlates with total mitochondrial Ca²⁺ load but not [Ca²⁺]_mito, perhaps through the formation of calcium-phosphate complexes.

In conclusion, understanding mitochondrial Ca²⁺ dynamics requires quantitative assessment of not only Ca²⁺ influx and efflux rates across the mitochondria, but rates of matrix Ca²⁺ buffering at different Ca²⁺ loads and rates of entry. The future development of computational models that incorporate all of these factors will help us understand these interesting features that are vitally important for Ca²⁺ homeostasis, cell death, and cardiac disease.