Mitochondria as all-round players of the calcium game

Abstract

Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca2+ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca2+, the possibility that physiological stimuli that raise the [Ca2+] of the cytoplasm could trigger major mitochondrial Ca2+ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca2+] rises. The direct measurement of mitochondrial [Ca2+] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca2+ signal is always paralleled by a much larger rise in [Ca2+] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca2+ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca2+ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca2+ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca2+ sources; (iii) the physiological consequences of Ca2+ transport for both mitochondrial function and the modulation of the cytoplasmic Ca2+ signal; and (iv) evidence that alterations of mitochondrial Ca2+ signalling may occur in pathophysiological conditions.

Figure 1. Schematic representation of the mitochondrial Ca²⁺ transport pathways in energised mitochondria

1, uptake pathways (uniporter, RaM). 2-4, efflux pathways: H⁺-Ca²⁺ (2) and Na⁺-Ca²⁺ (3) exchangers, and the permeability transition pore (4).

Figure 2. Agonist-dependent mitochondrial and cytoplasmic [Ca²⁺] increases in the presence and absence of the protonophore FCCP

The traces show calibrated [Ca²⁺] values obtained in HeLa cells transiently expressing an aequorin chimera localised in the mitochondria (A and C) or in the cytoplasm (B and D). Detection and calibration of the luminescence signals were carried out as previously described (Brini et al. 1995). Where indicated, HeLa cells were challenged with histamine (100 μm; Hist), an agonist coupled to the generation of inositol 1,4,5-trisphosphate and thus the release of Ca²⁺ from intracellular stores. In C and D, the cells were treated with 5 μm FCCP (carbonyl cyanide p-(trifluoro-methoxy)-phenylhydrazone), which was added 1 min before the agonist and maintained throughout the stimulation.

Figure 3. 3-D imaging of mitochondrial and ER structure in living HeLa cells

The two organelles were labelled with appropriately targeted chimeras of green fluorescent protein variants with different spectral properties. The images acquired at each focal plane were computationally deblurred and 3-D reconstructed. The mitochondrial and ER images are displayed in red and green, respectively, while the white areas are those in which overlap between the two images was detected. Reproduced with permission from Rizzuto et al. 1998.

Figure 4. Agonist-dependent mitochondrial [Ca²⁺] (A and C) and [ATP] (B and D) increases in the presence and absence of the ATP synthase inhibitor oligomycin

The calibrated [Ca²⁺] values were obtained as in Fig. 2, while ATP levels were inferred from light emission of a luciferase chimera targeted to the mitochondria (Jouaville et al. 1999). cps, counts per second. Where indicated, the cells were treated with 100 μm histamine. In C and D, the cells were treated with 2 μm oligomycin, which was added before the agonist and maintained throughout the stimulation.