Mitochondrial calcium homeostasis is primarily regulated by three pathways: the mitochondrial calcium uniporter (MCU), sodium/calcium/lithium exchanger (NCLX), and calcium hydrogen exchanger (CHE). The MCU is the dominant uptake pathway and is comprised of a heteromeric protein complex composed of various subunits including the mitochondrial Ca²⁺ uptake family (MICU 1, 2, and 3), essential MCU regulator (EMRE), MCU regulator 1 (MCUR1), MCU dominant-negative β–subunit (MCUb), and the solute carrier 25A23 (SLC25A23) [17]. These subunits form a complex on what appears to be an on-demand basis [17,18,19,20]. While an in–depth review on the molecular structure and function of these subunits are beyond the scope of this mini review/perspective, we would like to refer our readers to references [17,19,20]. Other pathways for Ca²⁺ uptake including the rapid mode calcium uptake and ryanodine receptors are also speculated to play an important role under certain conditions [21,22,23]. The MCU channel has a high affinity for Ca²⁺ (Kd ≤ 2 nM); however, it has a low half-activation constant (K_0.5~20 mM) [24]. As a result, the MCU is less active at lower concentrations of calcium (0.1–1 µM) and more active at higher extramitochondrial calcium loads [21]. The efflux pathways are primarily controlled by the NCLX and CHE. The NCLX is an electrogenic exchanger that swaps 3 Na⁺ (or Li⁺) for 1 Ca²⁺ [25,26]. Consequently, this reaction is electrogenic and sensitive to the mitochondrial membrane potential. Under physiological conditions, the CHE swaps calcium out for protons in the matrix in a manner that is presumed to be an electroneutral ratio of 1 Ca²⁺ per 2 H⁺ [27]. The calcium hydrogen exchanger functions independently of sodium and is present at much lower activities in tissues with high energy demand [28]. For instance, the CHE is dominant in the liver and other relatively quiescent tissues, while NCLX is predominant in the heart, brain, and other high activity tissues [29,30,31,32,33,34].

Under basal conditions, cytosolic Ca²⁺ concentrations are maintained in the 100 nM range [35]. For cardiomyocytes and skeletal myocytes, the range of global or average cytosolic Ca²⁺ concentrations during peak contraction that mitochondria are exposed to fall within 1 µM but can peak two to three times higher under stimulatory conditions [36]. That said, some mitochondria are exposed to higher concentrations (~10–100 µM) in microdomains associated with mitochondrial–SR contact sites [37,38,39]. In either the basal or stimulatory condition, intramitochondrial Ca²⁺ levels remain low as long as mitochondria remain coupled [37]. This form of calcium regulation is attributed to the mitochondrial calcium buffering system. What makes mitochondria particularly relevant in this scenario is their ability to store large amounts of calcium in their matrix [40].

This is of relevance in high–energy demand tissue as the mitochondrial membrane potential, Ca²⁺, and Na⁺ are the main regulators of mitochondrial calcium homeostasis. Additionally, while Ca²⁺ uptake is very sensitive to changes in membrane potential, Ca²⁺ efflux is less sensitive [41]. As a result, the MCU channel can load Ca²⁺ into the matrix at a rate far exceeding the NCLX matrix calcium clearance (1400 and 20 nmol Ca²⁺ min⁻¹ mg mitochondrial protein⁻¹, respectively) [42]. Hence, under conditions that disrupt cytosolic calcium homeostasis, Ca²⁺ uptake through MCU floods the matrix of energized mitochondria with massive amounts of Ca²⁺ [8]. Left unchecked, membrane potential loss precipitates a catastrophic collapse in energy homeostasis [2,43]. This phenomenon is often ascribed to the permeability transition phenomenon and has detrimental consequences for cell health and longevity [4,9,15,42,44]. This scenario places mitochondria in a vulnerable position, leading some to view the permeability transition phenomenon as a calcium overload release valve [45,46]. Regardless, the mitochondria Ca²⁺ uptake and removal processes are highly regulated with compensatory mechanisms in place to ensure cellular homeostasis and survivability. When such regulatory mechanisms fail, mitochondria become overloaded with calcium, and energy homeostasis collapses.

Calcium also influences mechanisms driving mitochondrial energy production and metabolic activity. For example, calcium regulates the activity of metabolic enzymes in the TCA cycle including pyruvate dehydrogenase, isocitrate dehydrogenase, and α–ketoglutarate dehydrogenase [2]. The TCA cycle generates reducing equivalents (such as NADH and UQH₂) used by the proton pumps that establish the membrane potential and alkalize the mitochondrial matrix relative to the cytoplasm [26]. This, in turn, biases ATP synthase away from its more favorable ATP hydrolysis set point to an operating regime conducive for ATP production via oxidative phosphorylation. Therefore, in high–energy demanding tissue where cells are constantly exposed to transient Ca²⁺ signals, calcium homeostasis is intrinsically linked to ATP production both through the TCA cycle and oxidative phosphorylation (oxphos) to regulate cellular bioenergetics.

Under most conditions, the membrane potential is the primary determinant of the ratio of matrix ATP to ADP and inorganic phosphate (i.e., the matrix ATP/ADP/Pi ratio). As Ca²⁺ is injected into mitochondria, matrix ATP/ADP ratios decline, the membrane potential depolarizes to a degree, and matrix pH increases [44,47]. As the matrix pH becomes more basic, dihydrogen phosphate (H₂PO₄⁻) is effectively driven into the mitochondria in symport with H⁺ in an electroneutral fashion. The phosphate carrier facilitates his symport, and when H₂PO₄⁻ enters the matrix, it undergoes a deprotonation event and forms HPO₄²⁻ [48]. Under these conditions, elevated Pi levels in the matrix facilitate the formation of calcium phosphate complexes. The formation of these complexes involves the deprotonation of HPO₄²⁻ into the phosphate trianion (PO₄³⁻) further releasing a H⁺. This helps counteract the significant alkalizing effect of accelerated proton pumping and charge replacement caused by Ca²⁺ uptake. Overall, a slight depolarization will alkalize the matrix pH which has the net effect of enhancing mitochondrial Ca²⁺ sequestration [43]. However, when the current generated by Ca²⁺ uptake exceeds the proton pumping current, thermodynamic driving forces reverse the F₁F_O ATP synthase activity and pumps protons out of the matrix via ATP hydrolysis [6]. When ATP is hydrolyzed from this reversal, the phosphate released can participate in phosphate precipitate formation until ATP is exhausted and the metabolic system collapses [40,49]. This is just one possible scenario as ATP hydrolysis is not a required source of inorganic phosphate during precipitate formation. In the presence of oligomycin, ATP synthase is inhibited, and mitochondria still possess the ability to take up massive amounts of Ca²⁺ when sufficient Pi is present [50]. Ultimately, when matrix Ca²⁺ concentrations exceed a threshold level, the formation of calcium phosphate precipitates in the matrix has the effect of reducing the mitochondrial free Ca²⁺ levels to manageable amounts via a type of buffering mechanism but at the expense of oxidative phosphorylation capacity [1,21].

We know that the consequences of precipitate formation operate on a spectrum (Figure 1), but we do not fully comprehend the mechanism. At low concentrations, calcium phosphate precipitates can have a protective effect. Whereas at high concentrations, precipitates can destabilize the mitochondrial cristae network [2,5]. This has been confirmed by others in which mitochondria loaded with Ca²⁺ resulted in calcium phosphate precipitates occupying more than 20% of the matrix volume [2]. One potential mechanism that leads to metabolic dysfunction is that precipitates may mechanically destabilize membrane structures by disrupting proteins involved in maintaining the cristae structure [5]. Another idea is that precipitates may serve as physical barriers limiting metabolite and substrate diffusion across the matrix [1]. The regulatory characteristics of the phosphate precipitation buffering mechanism remain enigmatic, but one concept boils down precipitate formation to a simple thermodynamic argument [40,49]. A second concept includes the idea that precipitate formation requires nucleation sites [51]. Full occupancy of these sites might dampen the extent of phosphate buffering within the mitochondrial matrix and send free Ca²⁺ high into pathological concentrations. The unknown nature of these potential nucleation sites makes it challenging to devise effective genetic and pharmacological approaches to manipulate mitochondrial calcium buffering. Thus, further study is required to resolve some of these unknowns.