Calcium Signaling: Double Duty for Calcium at the Mitochondrial Uniporter

Abstract

Uptake of Ca²⁺ by mitochondria serves as a regulator of a number of important cellular functions, including energy metabolism, cytoplasmic Ca²⁺ signals, and apoptosis. Recent findings reveal that the process of Ca²⁺ uptake by the mitochondrial uniporter is itself regulated by Ca²⁺ in a temporally complex manner.

The history of Ca²⁺ handling by mitochondria is a classic story of rags to riches (or rather riches to rags to riches) [1]. In the 1970s to early 1980s, mitochondria were thought to be prime sources of signaling Ca²⁺ in non-excitable cells. However, this idea fell into disfavor with the finding that, at the Ca²⁺ levels expected in either resting or activated cells, the relatively high Km for uptake should preclude significant Ca²⁺ accumulation by mitochondria [2], and ultimately with the finding that the signal for intracellular Ca²⁺ release, inositol trisphosphate (IP₃), clearly mobilized Ca²⁺ from endoplasmic reticulum [3]. Biochemical studies of mitochondrial Ca²⁺ content and the regulation of mitochondrial Ca²⁺-sensitive enzymes indicated that mitochondria were more likely to be a target for Ca²⁺ signaling rather than a source [4,5]. When Rizzuto et al. [6] made the first direct in situ measurements of mitochondrial Ca²⁺, it was clear that receptor-activated Ca²⁺ signals caused rapid and large Ca²⁺ signals in the mitochondrial matrix. It soon became apparent that mitochondria are capable of accumulating Ca²⁺ during signaling processes because they are positioned very near to either the sites of intracellular release (by IP₃, for example) or sites of entry across the plasma membrane (for example, through store-operated or voltage activated channels; for a review see [7]). Ca²⁺ activates several key enzymes in the mitochondrial matrix to enhance ATP production, and this provides an important mechanism for synchronizing energy production with the energy demands of Ca²⁺-activated processes during cell stimulation (excitation–metabolism coupling) [4,5,8]. In addition to serving as a target of Ca²⁺ signaling, the uptake of Ca²⁺ by mitochondria has important feedback effects to help shape cytosolic Ca²⁺ signals. This can occur through buffering of bulk cytosolic Ca²⁺ changes, but is most pronounced in the intimate intracellular ‘synaptic’ regions where mitochondria are in close proximity to Ca²⁺ release sites. Thus, rapid accumulation of Ca²⁺ can prevent or temper influences of Ca²⁺ on intracellular or plasma membrane channels [9,10]. Alternatively, in some instances mitochondrial Ca²⁺ uptake serves to compartmentalize Ca²⁺ signaling in appropriate cellular domains, a concept termed “firewall” from studies of pancreatic acinar cells [11]. In addition, under certain conditions, uptake of Ca²⁺ into mitochondria initiates a key step in the process of apoptosis through activation of the mitochondrial permeability transition pore, permitting escape of cytochrome c and other pro-apoptotic factors to the cytoplasm [12].

On the surface, mitochondrial Ca²⁺ handling seems rather simple. Uptake occurs through a channel termed a uniporter (from the Peter Mitchell nomenclature) and the rate of uptake depends upon driving force; this is considerable, as the process of electron transport in normally respiring mitochondria generates extremely negative transmembrane potentials across the inner mitochondrial membrane. This would ultimately lead to huge and potentially toxic levels of accumulated Ca²⁺ in the mitochondrial matrix were it not for the action of separate mitochondrial Ca²⁺ efflux pathways that are also coupled to the proton motive force developed by the respiratory chain. Thus, the inner mitochondrial membrane has a Ca²⁺/2H⁺ exchanger and/or a Ca²⁺/3Na⁺ exchanger analogous to that found in the plasma membrane. However, these efflux pathways can become saturated with high matrix Ca²⁺ loads, such that sustained rapid Ca²⁺ influx can still lead to mitochondrial Ca²⁺ overload.

In a report in a recent issue of Current Biology, Moreau et al. [13] reveal that the process of Ca²⁺ accumulation undergoes complex regulation by Ca²⁺ itself. They measured mitochondrial matrix Ca²⁺ concentration directly by loading the mitochondria of permeabilized mast cells (a rat basophilic leukemia line) with a fluorescent Ca²⁺ indicator. The uptake of Ca²⁺ was significantly reduced by inhibitors of calmodulin, suggesting that a Ca²⁺–calmodulin-mediated process is necessary for activation of the uniporter. This finding is consistent with an earlier observation that calmodulin antagonists impede the penetration of Mn²⁺ into mitochondria and that brief pulses of cytosolic Ca²⁺ can facilitate mitochondrial Ca²⁺ uptake [14]. Surprisingly, Moreau et al. [13] found that Ca²⁺ also appeared to inhibit its own uptake. Thus, uptake of Ca²⁺ due to addition of 100 μM Ca²⁺ was substantially impaired if preceded by exposure to 10 μM Ca²⁺. In contrast to the sensitization of mitochondrial Ca²⁺ uptake, the Ca²⁺-dependent inactivation was not sensitive to calmodulin blockers. The ability of Ca²⁺ to inactivate the uniporter may correspond to the phenomenon of desensitization of mitochondrial Ca²⁺ uptake suggested in earlier studies [15,16]. The uniporter appeared to be similarly sensitive to both activation and inhibition by Ca²⁺, with apparent Kds in the 10–20 μM range. This concentration range raises the standard mitochondrial problem of whether such Ca²⁺ concentrations are seen by the mitochondria during physiological signaling. In agreement with an earlier study [17], Moreau et al. show close associations between mitochondria and the endoplasmic reticulum, and go on to show that when Ca²⁺ is released from the endoplasmic reticulum by IP₃ there is a much more efficient transfer of Ca²⁺ to the mitochondria than that seen with exogenous Ca²⁺ addition. Thus, in the mast cell line, Ca²⁺ released by IP₃ is readily sensed by mitochondria, probably due to close apposition of release and uptake sites. There is ample evidence in the literature for such associations between mitochondria and the endoplasmic reticulum [18]. Interestingly, mitochondria have been shown to be somewhat mobile in cells, while the endoplasmic reticulum is relatively fixed; Ca²⁺ inhibits mitochondrial mobility, thus providing a mechanism to retain mitochondria at specific Ca²⁺ signaling sites [19].

The fact that both the activation and inactivation mechanisms have similar Ca²⁺ sensitivities would seem problematic for significant accumulation of Ca²⁺ in the mitochondrial matrix. However, when Moreau et al. [13] examined the kinetics of inactivation, it was found that inactivation occurred with a time constant of 17 seconds, while for uptake the time constant is about 6 seconds. This pattern of rapid activation and slower inactivation is reminiscent of myriad biological signaling patterns; most notably, as pointed out by Moreau et al. [13], of the biphasic regulation of Ca²⁺ discharge from the endoplasmic reticulum through the IP₃ receptor channel. In most cell types, physiological Ca²⁺ signaling occurs not by sustained elevations in cytoplasmic Ca²⁺, but through repetitive bursts of rising intracellular Ca²⁺, sometimes referred to as Ca²⁺ oscillations, with durations ranging from less than a second to several seconds [20]. The delayed inactivation mechanism will thus permit mitochondrial matrix Ca²⁺ to closely track these global signals, but will prevent excessive Ca²⁺ accumulation if the intracellular Ca²⁺ elevation is prolonged (Figure 1). For example, hormone-induced cytosolic Ca²⁺ oscillations in hepatocytes are closely paralleled by mitochondrial Ca²⁺ oscillations, whereas high levels of hormone that yield a sustained cytosolic Ca²⁺ increase cause only a transient spike of mitochondrial Ca²⁺ [8]. Thus, the Ca²⁺-dependent activation and inactivation of mitochondrial Ca²⁺ uptake [13] may help to tune mitochondrial responses to oscillatory Ca²⁺ signals, while filtering out persistent Ca²⁺ elevations that would have pathological consequences. Thus, once considered a simple Ca²⁺ accumulating depot, mitochondria have since proven to be elegantly controlled players in the complex network of intracellular Ca²⁺ signaling.

Figure 1.

Regulation of mitochondrial Ca²⁺ uptake.

Close apposition between mitochondria and endoplasmic reticulum (ER) Ca²⁺ release sites facilitates Ca²⁺ uptake into the mitochondria. A fast activation of the uniporter, mediated by calmodulin (CaM), enhances mitochondrial Ca²⁺ accumulation. A slower Ca²⁺-dependent inactivation of the uniporter serves to limit mitochondrial Ca²⁺ uptake during a prolonged rise of cytosolic Ca²⁺. The fast activation allows the mitochondrial matrix [Ca²⁺] to closely follow changes in the cytosolic [Ca²⁺], as occurs during cytosolic Ca²⁺ oscillations (left inset). The slower inactivation process limits mitochondrial Ca²⁺ uptake when the elevation of cytosolic Ca²⁺ is more sustained (right inset). (1) SERCA Ca²+ pump, (2) IP3 receptor Ca²⁺ release channel, (3) Ca²⁺ uniporter, (4) mitochondrial Ca²⁺/Na⁺ exchanger, (5) mitochondrial Ca²⁺/H⁺ exchanger, (6) ATP synthetase. The mitochondrial respiratory chain is shown as 3 cycles of proton pumping from the matrix.