Keywords
calcium oscillations, store-operated calcium entry, mitochondrial Ca2+ uniporter, mitochondrial Ca2+ uptake 1,
calcium oscillations, store-operated calcium entry, mitochondrial Ca2+ uniporter, mitochondrial Ca2+ uptake 1,
Most of the time, the hormone-induced Ca2+ increases that activate a variety of essential intracellular processes take the form of Ca2+ oscillations. In non-excitable cells, these repetitive spikes mainly arise through the periodic exchange of Ca2+ between the endoplasmic reticulum (ER) and the cytosol, through the interplay between inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channels and SERCA pumps1,2. This basic mechanism, summarized in Figure 1, has now been well characterized and accounts for the observed increase in the frequency of Ca2+ oscillations with increasing concentrations of InsP3 accompanying the rise in stimulation. Such a process is referred to as frequency encoding. It was often hypothesized that oscillations provide a digital signal to downstream effectors that are in turn stimulated in an ON or OFF manner. Indeed, if a process is activated above a threshold Ca2+ concentration, oscillations allow Ca2+ to reach this threshold repetitively even if the average Ca2+ signal remains below the threshold3,4.
Based on the observed frequency encoding of the extracellular signal, it was also postulated that the physiological response in the form of secretion, gene expression, proliferation, etc., would in turn be sensitive to the frequency of Ca2+ oscillations5–8. Although intuitively attractive, such frequency sensitivity of the downstream targets of Ca2+ has not been well corroborated by data. Besides the beautiful example of Ca2+-dependent calmodulin kinase II regulation by high-frequency Ca2+ spikes9 or of selective gene expression in T-lymphocytes4, there are few clear examples of physiological responses to Ca2+ increases that are quantitatively controlled by the frequency of the Ca2+ spikes. This statement does not mean that frequency encoding does not occur or that the frequency of Ca2+ oscillations does not affect the extent of the Ca2+-mediated physiological response. Indeed, a higher frequency of oscillations implies a larger average Ca2+ level, which may be per se the reason for the larger response. However, modulating the amplitude of the oscillations, their baseline level, or the duration of the spikes also modifies the average level and hence the response. As another example, spikes preceded by an important pacemaker-like Ca2+ increase could activate slower downstream targets characterized by a low threshold of activation. In such cases, frequency cannot be considered as the key characteristic of the oscillatory pattern and the response is not simply frequency sensitive. However, in the numerous studies about Ca2+ oscillations, frequency is the most studied parameter and the most commonly related to the extent of Ca2+-mediated physiological responses.
In fact, the relative scarcity of phenomena that are purely controlled by the frequency of Ca2+ oscillations is not so surprising given that the period of Ca2+ oscillations can be subject to a significant level of randomness (Figure 2 and 8,10). In some instances, it has even been explicitly observed that the frequency does not by itself regulate the extent of the second-messenger-mediated response. This is the case, for example, for carbachol-induced salivary secretion by acinar cells11. At mammalian fertilization, the total integrated Ca2+ signal input is the most relevant parameter ensuring completion of fertilization-associated events12. Interestingly, frequency encoding is also not a universal feature of Ca2+ oscillations, as it was shown in some cases, such as in acetylcholine-stimulated pancreatic acinar cells13, methacholine-stimulated lacrimal cells14, fish hepatocytes15, or in cell lines expressing the metabotropic glutamate receptor 516, that an increase in stimulation does not affect the frequency of the resulting Ca2+ oscillations. In these cases, of course, it cannot be expected that the frequency of Ca2+ oscillations would be the way by which cells encode the information related to the level of response that is precisely triggered by the stimulation.
Also, recent investigations tend to suggest that rather than the frequency alone, the detailed dynamic characteristics of the Ca2+ increase pattern play an important role in determining the extent of the cell response. As illustrated in Figure 2, in addition to frequency, Ca2+ oscillations can vary in the amplitude and the width of the spikes, the baseline Ca2+ level, and the degree of sustainability. We refer to modifications of one of these characteristics as fine tuning of Ca2+ oscillations to emphasize that they imply fine regulation of cytosolic Ca2+ that goes behind the mechanism schematized in Figure 1 accounting for the existence and the frequency of oscillations. Various observations corroborate that these properties are important determinants for the physiological response of the cell. For example, the CD147 factor promotes oncogenic activities and influences the progression of hepatocellular carcinoma by enhancing both the amplitude and the frequency of ER-dependent Ca2+ oscillations17. In intestinal stem cells, dietary and stress stimuli are integrated in such a way that frequent and robust Ca2+ oscillations are associated with a poised proliferative state, while smoother oscillations on a more elevated level accompany active proliferation18. Fine tuning of Ca2+ signals also plays a role in the differentiation of neuronal and muscle cells (see 19 for review). In astrocytes, knocking-down the Na+–Ca2+ exchanger (NCLX) that mediates Ca2+ release from mitochondria slightly affects cytosolic Ca2+ changes and, by doing so, significantly reduces Ca2+-dependent processes, such as glutamate release, wound closure, and proliferation20. Cell survival, death, and adaptation are sensitive to changes in Ca2+ patterns due to the interplay between ER/cytoplasmic Ca2+ exchanges and mitochondria and lysosomes21. Shigella bacteria also fine tune the Ca2+ responses when they invade epithelial cells. While the wild-type strain induces rather smooth and low-amplitude Ca2+ variations in the cytoplasm of the host cell, a less-invasive mutant strain induces more robust Ca2+ responses which, paradoxically, are associated with a higher survival of the host cells during the first hours following invasion22.
On another level, the precise isoforms of InsP3 receptors expressed by a given cell – which have been shown to substantially affect the shape of the Ca2+ oscillations23–25 – are critical for cell death and survival decisions26. Finally, bioinformatics analyses highlighted that in cancer cells and tissues, the main processes associated with Ca2+ dynamics that are perturbed are the mechanisms of store-operated calcium entry (SOCE) and of calcium reuptake into mitochondria27. Both of these processes are related to the fine tuning of Ca2+ oscillations, as discussed below. Altogether, these observations call for a more detailed understanding of oscillation-associated Ca2+ dynamics. Understanding why Ca2+ oscillates and what regulates the frequency of oscillations is not sufficient to understand their physiological impact, but the duration and shape of the peaks, their sustainability, and the baseline Ca2+ level must be carefully taken into account. In the following sections, we elaborate on two key controllers of the InsP3R-based Ca2+ oscillations, both related to Ca2+ stores other than the ER, namely the mitochondria and the extracellular medium. We briefly review and discuss some of the main recent observations about their interplay with the InsP3-induced Ca2+ spikes.
By stimulating the activity of key enzymes involved in mitochondrial ATP synthesis, Ca2+ entry into mitochondria stimulates metabolism, thereby coupling ATP synthesis with energy demand28. That Ca2+ exchange between the cytosol and the mitochondria in turn affects InsP3-induced cytosolic Ca2+ signals was put forward quite early29,30, but this concept was somewhat put aside for a decade. The molecular identification of the mitochondrial Ca2+ uniporter (MCU), a voltage, cytosolic, and mitochondrial Ca2+-sensitive transporter31–34, awakened interest in this question. Ca2+ entry into mitochondria through the MCU is a highly non-linear function of cytosolic Ca2+33. The MCU is in fact the Ca2+ pore-forming component of the uniporter and is part of a large complex of proteins that are required for Ca2+ channel activity or to regulate it under various conditions. For example, MICU1 (mitochondrial Ca2+ uptake 1) limits mitochondrial Ca2+ influx at low cytosolic Ca2+ concentration and the interaction between the MCU and MICU1 requires the expression of another component, called EMRE for essential MCU regulator34,35. Ca2+ efflux back into the cytoplasm occurs through a NCLX. As expected, modifying any of these pathways affects the frequency of the oscillations; interestingly, increasing the activity of the MCU can both increase and decrease the frequency of oscillations36. In addition to its effect on the frequency of the oscillations, the MCU controls the width of the spikes and the sustainability of the oscillations, as knocking-down the MCU broadens Ca2+ oscillations and accelerates the rundown of the oscillations in rat basophilic leukemia (RBL)-1 cells (Figure 3). Such rundown suppresses gene expression in response to leukotriene receptor activation37. Mitochondria also affect the rate of rise and fall of cytosolic Ca2+ and thus the half-width and duration of the spike. More specifically, mitochondria smooth out cytosolic Ca2+ changes mainly because they have a ~30 times larger Ca2+ buffering capacity than the cytoplasm38. Also, because of their slow dynamics, mitochondria continue releasing Ca2+ between subsequent releases of Ca2+ from the ER, thus playing a key role in determining the baseline cytosolic Ca2+ level. Thus, mitochondrial Ca2+ handling through the MCU and the NCLX clearly fine tunes cytosolic Ca2+ oscillations.
The kinetics of the MCU and the NCLX have been fairly well characterized, but much remains to be done to fully identify other fluxes. The permeability transition pore (PTP) in its low conduction mode participates in the Ca2+ exchange process in HeLa cells, as its inhibition by cyclosporine A affects Ca2+ oscillations36,39. The functional role of LETM1-mediated Ca2+ transport also remains poorly understood. This EF-containing transmembrane protein has been functionally identified as a Ca2+/H+ exchanger of the inner mitochondrial membrane40,41, although this remains controversial42. In electrically excitable cells such as cardiomyocytes and neurons, ryanodine receptors have been shown to transport Ca2+ into mitochondria43,44. A rapid Ca2+ uptake mode (RaM) of poorly identified molecular nature has been reported in studies on isolated mitochondria from cardiac and liver cells45,46. However, the implication of RyR and RaM in mitochondrial Ca2+ influx remains to be firmly established47,48. Finally, in a more indirect manner, mitochondrial metabolism also affects cytosolic Ca2+ signals, mainly by modifying the mitochondrial voltage across the internal mitochondrial membrane, which greatly affects the activities of all of the above-mentioned fluxes29. All of the above-cited phenomena are thus potentially implicated in the control of the detailed characteristics of Ca2+ oscillations. Their interplay with the activities of the MCU and the NCLX is regulated by an intricate and complex network of interactions implicating cytosolic and mitochondrial Ca2+ as well as mitochondrial voltage and numerous accessory proteins.
Cytosolic Ca2+ oscillations are sustained by SOCE from the extracellular medium49. This mechanism involves the stromal interaction molecule (STIM) and the Orai protein50. The transmembrane ER protein STIM is sensitive to Ca2+ changes in the ER through an EF-hand facing the lumen of the store. Decrease in luminal Ca2+ below ~200 μM (for the STIM1 isoform) leads to STIM aggregation, followed by migration to ER–plasma membrane junctions. Here, STIM oligomers can bind and activate Orai, a four-transmembrane-domain plasma-membrane-spanning protein, thus forming a channel (known as CRAC for Ca2+-release-activated Ca2+ channel) allowing Ca2+ to enter down the chemical gradient. Another STIM isoform, STIM2, has a lower affinity for ER Ca2+, which allows for activation of Ca2+ entry at moderate ER depletion, although at a reduced rate51. Mammalian cells have genes for the three homologs Orai1, Orai2, and Orai3, and it is thought that Orai2 and/or Orai3 act as compensative types for the lack of Orai1. Orai channels are made of multiple subunits, and CRAC channel gating by STIM is best described by a Monod-Wyman-Changeux scheme in which tetramers of Orai have four STIM binding sites50,52.
Although the mechanism just described has most of the time been investigated in conditions when the Ca2+ pools are emptied artificially, studies performed in a variety of cell types demonstrate that STIM expression is essential for an ensemble of physiological processes53. To quote here just one recent example, in airway smooth muscle, altered expression and function of STIM/Orai proteins have been linked to pathologies including restenosis, hypertension, and atopic asthma54.
The STIM-Orai pathway for Ca2+ entry displays a hysteretic behavior: STIM-Orai association and dissociation do not occur at similar ER Ca2+ concentrations55. Although the origin and the physiological significance of this unusual behavior remains unknown, it might be related to the inactivation of SOCE-mediated Ca2+ entry by cytosolic Ca2+ itself, a process that has long been thought to be mediated by calmodulin56 but was recently suggested to be due to a calmodulin-independent conformational change within the pore allowed by two specific Orai residues, Y80 and W7657,58. This Ca2+-induced inactivation (CDI) of SOCE allows for a modulation of Ca2+ entry depending on the level of cytosolic Ca2+, thus shaping the oscillations.
The key effect of STIM and Orai on the oscillatory Ca2+ pattern and its downstream targets are also much documented. Through the specific ER Ca2+ sensor STIM2 that has a high KM for Ca2+, SOCE determines the basal level of Ca2+ in HeLa cells51. More generally, SOCE-mediated Ca2+ entry has a significant effect on Ca2+ oscillations, as it can in turn affect all Ca2+ exchanges between the cytoplasm and the internal stores. A less straightforward but highly interesting effect was uncovered in RBL-2H3 cells. Because the activity of the plasma membrane phosphatidylinositol 4-phosphate 5 kinase that replenishes the PIP2 pool is Ca2+ sensitive, SOCE is necessary to avoid the rundown of the oscillations. Indeed, in the absence of SOCE, cysteinyl leukotriene type I receptor activation leads to the exhaustion of the PIP2 pool and hence to the disappearance of InsP3-induced Ca2+ release from internal stores59.
In RBL cells displaying Ca2+ oscillating, gene expression is entirely driven by SOCE and proceeds as an all-or-nothing process in individual cells60. During maturation of mouse oocytes, STIM1 and Orai1 control the basal Ca2+ level and the whole Ca2+ homeostasis, thus controlling meiosis resumption61. At fertilization of pig eggs, overexpression of STIM1 and Orai1 substantially decreases the number of Ca2+ spikes induced by sperm binding (Figure 4). Moreover, these spikes are broader and their frequency is reduced as compared to control eggs62. This observation contrasts with the observed decreased frequency of fertilization-induced Ca2+ oscillations in hamster eggs when decreasing external Ca2+ concentration63. It shows that the control of Ca2+ signaling by SOCE cannot be directly assimilated to the control of Ca2+ signals by the extracellular Ca2+ concentration. Interestingly, if SOCE is inhibited, fertilization is also impaired, as oscillations last for about 1 hour instead of at least 2 hours. In mice, cytoplasmic Ca2+ levels are elevated for ~50% of the time in STIM1+Orai1-overexpressing oocytes in the first 2 hours after fertilization, as compared to only less than 20% of the time in control oocytes. Despite this larger Ca2+ signal, most of the STIM1/Orai1-overexpressing oocytes do not reach the two-cell stage64. However, female mice lacking Orai1 are fertile65, while male mice are sterile due to severe defects in spermatogenesis66.
It is by now clear that in many cases the existence of Ca2+ oscillations does not provide an ON/OFF signal for the Ca2+-mediated response to the stimulus nor is the extent of the response only determined by the frequency of the oscillations. How the exact shape of this Ca2+ signal is controlled, i.e. what we refer to here as its fine tuning, can alter the response qualitatively and quantitatively. Ca2+ exchanges with mitochondria and SOCE play an important role in fine tuning cytosolic Ca2+ oscillations. Interestingly, there is some dynamic interplay between these two Ca2+ sources as, in mesothelial cells in the absence of external Ca2+, mitochondrial Ca2+ takes over to provide a Ca2+ influx pathway during oscillations67. Moreover, other organelles such as the Golgi68,69 or the acidic organelles21,70,71 are also involved. Genetic regulations further complicate the intricate network of Ca2+ fluxes: in lymphocytes, Ca2+-dependent activation of CREB controls the level of expression of the MCU, which explains why the expression of this uniporter is modified in the absence of InsP3 receptors or of the STIM/Orai machinery72. Much remains to be done to understand how diverse factors interact to control the detailed pattern of Ca2+ oscillations and how this pattern can in some cases significantly affect the physiological response. Integration of the Ca2+ signal over long periods of time may explain why small changes in the pattern of the Ca2+ spikes become significant in some cases. By such integration, the extent of activation of the downstream targets of Ca2+ is modified by apparently minor changes in the Ca2+ oscillatory pattern, which are less visible than its frequency. Spatial aspects most certainly also play an important role in this respect, as the Ca2+-sensitive targets are far from being homogeneously distributed within the cell73. Finally, the kinetics and thresholds for Ca2+ activation of these targets are expected to be at least as important, as in other signaling cascades playing a key role in the storage of information74.
Geneviève Dupont is Research Director at the Belgian FRS-FNRS. This work was supported by the FNRS CDR n° J.0007.1.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
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