Shaped by leaky ER: Homeostatic Ca2+ fluxes

At any moment in time, cells coordinate and balance their calcium ion (Ca²⁺) fluxes. The term ‘Ca²⁺ homeostasis’ suggests that balancing resting Ca²⁺ levels is a rather static process. However, direct ER Ca²⁺ imaging shows that resting Ca²⁺ levels are maintained by surprisingly dynamic Ca²⁺ fluxes between the ER Ca²⁺ store, the cytosol, and the extracellular space. The data show that the ER Ca²⁺ leak, continuously fed by the high-energy consuming SERCA, is a fundamental driver of resting Ca²⁺ dynamics. Based on simplistic Ca²⁺ toolkit models, we discuss how the ER Ca²⁺ leak could contribute to evolutionarily conserved Ca²⁺ phenomena such as Ca²⁺ entry, ER Ca²⁺ release, and Ca²⁺ oscillations.

Introduction

The tight control of coordinated homeostatic calcium ion (Ca²⁺) fluxes is of fundamental importance for cellular signaling and health (Berridge et al., 2003). Evolutionary conserved mechanisms maintain Ca²⁺ levels in every cell type, while different adaptions modulate cell type-specific functions. The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER) (Verkhratsky, 2005). Despite its central role in the pathophysiology of many severe diseases (Mekahli et al., 2011), our understanding of how ER Ca²⁺ fluxes shape cellular Ca²⁺ signaling is still poor. One reason is that Ca²⁺ signals are typically monitored in the cytosol. With the improvement of ER Ca²⁺ imaging techniques, it became technically possible to directly monitor Ca²⁺ dynamics in the ER, with reasonably good spatiotemporal resolution (Rehberg et al., 2008; Samtleben et al., 2013; Rodriguez-Garcia et al., 2014; de Juan-Sanz et al., 2017; Schulte et al., 2022). These experiments unraveled a surprisingly pronounced physiological role of the ER Ca²⁺ leak in shaping Ca²⁺ signals (Thastrup et al., 1989; Camello et al., 2002; Flourakis et al., 2006; Samtleben et al., 2015; Lemos et al., 2021).

Principles and limitations of ER Ca²⁺ imaging

In the cytosol, the resting Ca²⁺-concentration is about 100 nM. Upon stimulation, cytosolic Ca²⁺ concentrations rise to 0.5–1 µM and can reach tens of micromolar close to active Ca²⁺-channels (Bootman and Bultynck, 2020). In the ER lumen, Ca²⁺ concentrations are in the range of ∼50 µM up to 1 mM. For this reason, ER Ca²⁺ indicators need a low affinity for Ca²⁺ [dissociation constant (K_d) ∼100–200 µM] while maintaining high responsiveness to Ca²⁺.

For ER Ca²⁺ imaging, three fundamentally different principles were developed. One is based on the direct loading of cells with synthetic acetoxymethyl (AM)-estered Ca²⁺ indicators, such as Mag-Fura2-AM [Kd ∼ 25–50 µM (Hofer and Machen, 1993; Solovyova and Verkhratsky, 2002)], Mag-Fluo4 [K_d ∼ 22 μM (Laude et al., 2005)] or Fluo5N [∼90 μM (Chen et al., 2015)]. The technique is well-suited for some cell types; however, a certain amount of the indicator becomes reactive in the cytosol, thereby causing ‘mixed’ ER-cytosol signals. To remove undesirable indicator from the cytosol, cells can be permeabilized (e.g., with digitonin or streptolysin) or dialyzed with the help of a patch-clamp pipette (Solovyova and Verkhratsky, 2002). In permeabilized cells, Mag-Fluo4 leaking out of the ER can also be quenched with an antibody (Rossi and Taylor, 2020). Surprisingly, after quenching, Mag-Fluo4-AM showed a much higher K_d^Ca2+-value in the ER (∼1 mM instead of 22 µM), most likely due to incomplete de-esterification in the ER lumen (Rossi and Taylor, 2020).

A strategy to accumulate synthetic Ca²⁺ indicators in the ER lumen of non-disrupted cells is targeted esterase-induced dye loading (TED) (Rehberg et al., 2008; Samtleben et al., 2013). For TED, a genetically overexpressed carboxylesterase hydrolyses a synthetic low-affinity acetoxymethyl (AM) ester in the ER lumen, thereby forming a hydrophilic dye/Ca²⁺ complex. The fluorescent Ca²⁺-dye complex is trapped and enriched in the ER lumen and provides an excellent signal-to-noise ratio (Rehberg et al., 2008). The best available indicator for TED is still Fluo5N-AM (Samtleben et al., 2013). The de-estered, Ca²⁺-sensitive form of Fluo5N is detectable in the ER for hours. In the cytosol, Fluo5N is reactive but barely visible. Unfortunately, Fluo5N is extremely light sensitive (bleaching and random flashing), making it difficult to image Fluo5N/Ca²⁺ complexes (Samtleben et al., 2013; Schulte et al., 2022).

The third strategy is based on ER-targeted low-affinity GECIs (genetically encoded Ca²⁺ indicator) such as the D1ER-derivate D4ER (Kipanyula et al., 2012), CEPIAer (Suzuki et al., 2014), ER-GCaMP6-150/210 (de Juan-Sanz et al., 2017) or ER-GAP-derivates (Rodriguez-Garcia et al., 2014; Alonso et al., 2017). For GECIs, high expression levels using a strong vector promoter are required to achieve an appropriate GECI signal. This increases the risk of protein misfolding or mistargeting by saturating the ER translocation and ER retention and retrieval processes.

We recently compared TED using Fluo5N with the GECI ER-GCaMP6-150. The data showed that TED is well suited to visualize fast Ca²⁺ signal onsets (Schulte et al., 2022). ER-GCaMP6-150 showed excellent on-off rates, was quite bleach resistant and allowed imaging for up to 1 h on the same cells (Schulte et al., 2022). In all our direct ER imaging experiments, ‘typical’ excitation light conditions could stop ongoing ER Ca²⁺ oscillations within ∼2 min (Schulte et al., 2022), albeit the indicators themselves were still reactive. We observed the phenomenon, loss of reactivity, in all types of cells we ever investigated (Hek293, HeLa, BHK21, astrocytes, neurons). We do not have an explanation for this observation. Hence, extreme low excitation light conditions might be needed for all ER Ca²⁺ imaging experiments as the light sensitivity of ER Ca²⁺ dynamics might be of biological and not methodological origin.

Nowadays, for dual-color Ca²⁺ imaging (ER/cytosol), a green-fluorescent ER Ca²⁺ indicator and a red fluorescent cytosolic dye are a good combination (Rodríguez-Prados et al., 2020; Schulte et al., 2022). We recommend using a GECI, such as ER-GCaMP6-150/210, with AM-ester based loading of the cytosolic dye Cal-590 (Birkner and Konnerth, 2019; Schulte et al., 2022). Fluorescence of both dyes can be well separated with standard fluorescence microscopy. The excellent signal-to-noise ratio of Cal-590 allows low-light illumination conditions and does not destroy ER Ca²⁺ dynamics (Schulte et al., 2022).

Is there a defined ‘resting’ ER Ca²⁺ concentration?

In physiology, the extracellular and cytosolic ion concentrations are well defined. This is not true for the ER Ca²⁺ concentration. Resting ER Ca²⁺ levels were described to be in the range of 50 µM up to 1 mM; meaning a difference factor of ×20. Depending on cell type, indicator, or calibration approach, resting ER Ca²⁺ concentrations range between 60 and 270 µM in cultured sensory neurons (Solovyova et al., 2002), 700–800 µM in HeLa and Hek293 cells (Tang et al., 2011), ∼150 µM in primary hippocampal neurons (de Juan-Sanz et al., 2017), and ∼400 µM in cultured astrocytes (Rodríguez-Prados et al., 2020).

It is not easy to determine the exact ‘resting’ ER Ca²⁺ concentration in living cells. The ER Ca²⁺ range can be estimated in permeabilized [‘leaky cells’ (Streb et al., 1983)] or disrupted [‘whole-cell patch clamp (Solovyova et al., 2002)] conditions, after blockade of the SERCA. Standardized Ca²⁺ calibration solutions (zero Ca²⁺ to ∼2–5 mM free Ca²⁺) are applied extracellularly until an equilibrium state is formed between the extracellular space, the cytosol, and the ER lumen. However, permeabilized cells are no longer in a physiological state, and potential loss of small-molecule Ca²⁺ dyes may confound Ca²⁺ calibration. Also, it is difficult to provide a ‘resting’ ER Ca²⁺ level for living cells with unknown Ca²⁺ toolkit and physiological state.

We imaged an entire calibration process for ∼20 min with a temporal resolution of 5 Hz (Schulte et al., 2022). The data confirmed that Ca²⁺ concentration in the ER lumen is about thousand-fold higher than in the cytosol and about 10–20x times lower than in the extracellular space (Schulte et al., 2022). Notably, the ER Ca²⁺ store can be rapidly refilled, within seconds, when the SERCA is blocked (Schulte et al., 2022). It can well be that this fast passive ER refilling in permeabilized cells is a ‘calcium tunnelling’ phenomenon (Petersen et al., 2017) and occurs through the ER Ca²⁺ leak channels.

The ER Ca²⁺ leak, a surprisingly strong intracellular Ca²⁺ flux

In a simplified view, a living cell generates the resting membrane potential by potassium ions that leak from inside the cell to the outside, via K⁺ ‘leak’ channels. The driving force of the potassium gradient is maintained by the high energy consuming Na⁺/K⁺ ATPase. The fundamental principle is similar for Ca²⁺ fluxes from the ER Ca²⁺ store to the cytosol. A very high force drives the Ca²⁺ from the ER lumen through the ER Ca²⁺ leak channels into the cytosol, an effect which might be electrogenic (Burdakov et al., 2005; Verkhratsky, 2005). The molecular identity of the ER Ca²⁺ leak is not entirely clear (Lemos et al., 2021), but there is strong experimental evidence that the Sec61 translocon complex is one of the main mediators of passive ER Ca²⁺ leak (Flourakis et al., 2006; Schäuble et al., 2012). Sec61 complexes are evolutionarily highly conserved, are ubiquitous, and transcriptome data revealed that they are expressed at very high levels. Sec61 complexes are non-redundant proteins involved in protein synthesis (Lang et al., 2017), meaning that a cell cannot fully avoid ER Ca²⁺ leak. To maintain ER Ca²⁺ levels, high activity of the SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase) is needed.

An easy way to unmask the ER Ca²⁺ leak is by blocking the SERCA, irreversibly with the drug thapsigargin (Thastrup et al., 1989), or acutely with CPA (cyclopiazonic acid) (Samtleben et al., 2015). The rate at which SERCA blockade empties the ER can vary widely from cell to cell. Direct ER Ca²⁺ imaging, however, suggests that the ER Ca²⁺ leak is a strong, temperature-dependent, persistent intracellular Ca²⁺ flux. In neurons, for instance, SERCA blockade with CPA depletes the ER Ca²⁺ store within 1–2 min (Samtleben et al., 2015; de Juan-Sanz et al., 2017). In cultured astrocytes, acute application of SERCA blocking agents [thapsigargin (Schulte et al., 2022) or tBHQ (Rodriguez-Prados et al., 2020)] caused a drop in the fluorescence signal (F) from F_rest to F_min in about a minute.

Ca²⁺-imaging in the cytosol is not well-suited to investigate the spatio-temporal dynamics of the ER Ca²⁺ leak. In neurons, in presence of extracellular Ca²⁺, SERCA blockade induces Ca²⁺ entry over the plasma membrane, thus masking the contribution of the ER Ca²⁺ leak to the cytosolic Ca²⁺ transient. In Ca²⁺-free extracellular solution, the expected cytosolic Ca²⁺ signal is often barely detectable, because both the ER and cytosolic Ca²⁺ are rapidly lost to extracellular sites (Samtleben et al., 2015).

The ER Ca²⁺ leak triggers homeostatic Ca²⁺ fluxes

Existence of an evolutionarily conserved ER Ca²⁺ leak raises the question of its influence on homeostatic Ca²⁺ fluxes (minimal model in Figure 1A). When we blocked neuronal activity of hippocampal neurons and removed extracellular Ca²⁺ acutely, the ER Ca²⁺ signal dropped to a F_min plateau signal within a few minutes (Figure 1B) (Samtleben et al., 2015). Subsequent acute addition of CPA did not further reduce the ER Ca²⁺ signal. Similarly, metabotropic ER Ca²⁺ release was abolished in cerebellar Purkinje cells that were kept in Ca²⁺-free solution for some minutes, as shown with cytosolic Ca²⁺ imaging (Hartmann et al., 2014). Removal of extracellular Ca²⁺ appear to empty the ER Ca²⁺ store virtually completely in some minutes.

FIGURE 1

FIGURE 1. ER Ca²⁺ leak in neurons triggers resting Ca²⁺ entry. (A) Minimal model: ER Ca²⁺ leak and cellular Ca²⁺ loss (green) need to be counterbalanced from extracellular sides (orange). Activity of the SERCA closes the Ca²⁺ loop. Leaking Ca²⁺ is partly rescued from the cytosol by the SERCA, but this cannot prevent ER Ca²⁺ depletion in Ca²⁺-free solution. The best candidates for homeostatic Ca²⁺ influx are ORAI channels and yet unknown Ca²⁺ entry (CE?) mediators. (B) During extracellular Ca²⁺ withdrawal, ER Ca²⁺ levels start to drop within seconds. Extracellular Ca²⁺ is needed to counterbalance homeostatic ER Ca²⁺ loss. (C) Inhibition of Ca²⁺ entry by short application of the SOCE-inhibitor SKF-96365 (25 µM) reduces ER Ca²⁺ levels. (D) ER Ca²⁺ refilling with and without acute SERCA blockade. A short, transient ER Ca²⁺ signal is observed in presence of the SERCA inhibitor (indicated by two black lines). The original experiments were performed in primary hippocampal neurons, during neuronal activity blockade with 100 nM tetrodotoxin. Cells were not stimulated via any receptor (R). Cytosolic Ca²⁺ signals were measured with Oregon Green 488 BAPTA-1 and are shown in magenta. TED with Fluo5N-AM were used for direct ER Ca²⁺ imaging (green lines in A–C). [Modified according to (Samtleben et al., 2015)].

Evidently, passive ER Ca²⁺ loss cannot be restored from cytosolic calcium by SERCA activity alone. Hence, a constitutively active resting Ca²⁺ entry is needed to maintain ER Ca²⁺ levels. The phenomenon in which depletion of the intracellular Ca²⁺-store activates Ca²⁺ influx is called store-operated Ca²⁺ entry (capacitive Ca²⁺ entry) (Putney et al., 2017). We tested SOCE-blockers to find out whether a constitutively active calcium entry mechanism compensates passive ER Ca²⁺ loss. The data confirmed that acute application of SOCE-blockers (SKF-96365/BTP-2) induces an immediate drop in ER Ca²⁺ levels (Figure 1C) (Samtleben et al., 2015). Thus, resting Ca²⁺ influx over the plasma membrane exists and is, in the end, triggered by the ER Ca²⁺ leak and maintained by SERCA activity (summarized in Figure 1A).

The resting Ca²⁺ influx is functionally relevant as it is a distinct mechanism for regulating gene expression (Lalonde et al., 2014) and seem to also trigger local Ca²⁺ influx events, so-called ‘signal-close-to-noise Ca²⁺ activity’ (Prada et al., 2018). The Ca²⁺ toolkit underlying homeostatic Ca²⁺ influx mechanisms is not well known (Figure 1A) but in hippocampal neurons, it is resistant to an inhibitor cocktail containing TTX (for voltage-gated sodium channels), APV and CNQX (to block ionotropic glutamate receptors), and Ni²⁺-ions (to reduce activity of low-threshold activated VGCCs) (Prada et al., 2018). We think that constitutive active ORAI channels contribute to ER-leak-triggered, homeostatic Ca²⁺ influx (Figure 1A).

Why are neurons or astrocytes investing so much energy in maintaining homeostatic Ca²⁺ fluxes via the extracellular space? Perhaps, resting Ca²⁺ fluxes are needed to signal neuronal health. More SOCE-like Ca²⁺ entry or less active removal of cytosolic Ca²⁺ would lead to cellular Ca²⁺ overload. This has clinical implications. For instance, when SOCE blockers are used to prevent acute or neurodegenerative Ca²⁺ overload, resting homeostatic Ca²⁺ influx would be reduced. This would also reduce ER Ca²⁺ levels and thereby induce ER stress signaling (Mekahli et al., 2011) and mitochondrial dysfunction (Garbincius and Elrod, 2022).

SERCA-independent ER refilling

Theoretically, fast passive Ca²⁺ influx from the extracellular side might be enough to locally refill the ER lumen. In a direct ER Ca²⁺ imaging experiment, we emptied the ER Ca²⁺ store of neurons in Ca²⁺-free solution (Figure 1D). When we re-added extracellular Ca²⁺ and blocked the SERCA acutely, a short, transient increase in ER Ca²⁺ levels was observed (Samtleben et al., 2015). We cannot exclude incomplete block of the SERCA in this experiment. However, the transient-like character of the signal suggests that Ca²⁺ enters the ER passively and is then lost through the ER Ca²⁺ leak. Future developments in life-cell imaging combined with super-resolution techniques might solve the question whether there are regulated ‘tunnel-like’ microdomains between the ER lumen and the extracellular space. General models for Ca²⁺ tunnelling mechanisms are discussed since many years (Petersen et al., 2017). It can well be that ER Ca²⁺ sparks (Cheng and Lederer, 2008) depend on such a ‘tunnel-like’ microdomain. Proximity of ‘leaky’ ER microdomains, ORAI, and Stim complexes might be a minimal requirement for electrogenic, local Ca²⁺ signals. The fluxes should be sufficient to trigger voltage-dependent Ca²⁺ influx as well as local induction of Ca²⁺-induced Ca²⁺ release (Ca²⁺-iCR) (see later).

Shaping of Ca²⁺ fluxes by the ER Ca²⁺ leak: Clues from cultured astrocytes

In our recent work, we used cultured cortical astrocytes (mouse) and dual color Ca²⁺ imaging (ER/cytosol) to find out how ER Ca²⁺ dynamics shape homeostatic Ca²⁺ fluxes. Astrocytes are well suited as a prototypical Ca²⁺ model (Verkhratsky and Nedergaard, 2018; Lim et al., 2021). The cells are very responsive and can be cultured with high purity, which makes it easy to determine their Ca²⁺ toolkit, e.g., with RNA-seq (Hasel et al., 2017; Schulte et al., 2022). The cell model expresses two IP₃ receptors (Itpr1 and Itpr2), but no ryanodine receptors. Notably, there is just one SERCA (SERCA2, Atp2a2) and a high amount of plasma membrane Ca²⁺-ATPases (Atp2b1, Atp2b4). Sec61a and other ER Ca²⁺ leak channel candidates (e.g., Tmco1, presenilin) are highly expressed (Schulte et al., 2022). Based on transcriptome data and analysis of calcium signal profiles observed in individual cells, minimal models can be designed to discuss how the ER Ca²⁺ leak could shape Ca²⁺ signals.

The ER Ca²⁺ leak is interlinked with Ca²⁺-induced Ca²⁺ release

Cultured cortical astrocytes show depolarization-dependent ER Ca²⁺ release (Rodríguez-Prados et al., 2020), but do not express ryanodine receptors (Hasel et al., 2017; Schulte et al., 2022). Surprisingly, ER Ca²⁺ refilling by Ca²⁺ entry can induce Ca²⁺-iCR, which empties the ER Ca²⁺ store again, and keeps cytosolic Ca²⁺ levels high (Figure 2A) (Schulte et al., 2022). In astrocytes, the effect was observed: 1) after adenosine-induced Ca²⁺ release in Ca²⁺-free solution and delayed re-adding of extracellular Ca²⁺ (Figure 2A, middle panel); 2) during ER replenishment by homeostatic Ca²⁺ entry after depleting the ER Ca²⁺ store in Ca²⁺-free solution (Figure 2A, lower panel; for neurons see Figure 1B). The effect might be mediated by IP₃-receptors, or ER Ca²⁺ leak channels by a yet unknown mechanism. Returning to resting ER Ca²⁺ levels would then require cytosolic Ca²⁺ export to extracellular sides, e.g. by active transport via Ca²⁺-ATPases or secondary active transport mechanisms (Na⁺/Ca²⁺ exchanger).

FIGURE 2

FIGURE 2. Induced ER Ca²⁺ release versus ER Ca²⁺ oscillation in cultured astrocytes. Simultaneous imaging of ER and cytosolic Ca²⁺ with the indicators: ER-GCaMP6-150 (green) and Cal-590-AM (magenta) in non-disrupted cells. (A) Minimal model: Ca²⁺-induced Ca²⁺ release (Ca²⁺-iCR, cyan) is induced by SOCE-like Ca²⁺ entry after ER replenishment. Middle panel: In Ca²⁺-free solution, adenosine-induced Ca²⁺ oscillations persist for about half a minute. Delayed Ca²⁺ replenishment induces a Ca²⁺-iCR phenomenon, in absence of ryanodine receptors (cyan arrow). Lower panel: Removal of extracellular Ca²⁺ and subsequent re-adding of extracellular Ca²⁺ can be sufficient to stimulate Ca²⁺-iCR. (B) (Minimal model) Spontaneous Ca²⁺ oscillation in Ca²⁺-free solution. (Middle panel) Spontaneous Ca²⁺ oscillations are shaped by a circular relationship between ER and cytosolic signals. Simultaneous imaging revealed a time lag of some seconds between peak signals in the cytosol and the ER Ca²⁺ release signal (grey). Raw traces (black dashed line) and the low-pass filtered traces (in color) are plotted. Lower panel: Spontaneous Ca²⁺ oscillation in Ca²⁺-free solution. The sarco-/endoplasmic reticulum Ca²⁺ ATPase (SERCA) recycles intracellular Ca²⁺. Candidates for ER Ca²⁺ leak are IP₃ receptors and ER Ca²⁺ leak channels. Ca²⁺ released from the ER stays in the cell, is not exported to extracellular sides (compare with Figure 3), and the SERCA adapts its activity to the cytosolic Ca²⁺ concentration. [Modified according to (Schulte et al., 2022)].

Is the ER Ca²⁺ leak the basic trigger for spontaneous Ca²⁺ oscillations?

Ca²⁺ oscillations in astrocytes can also appear spontaneously, without an obvious external stimulator. Ca²⁺ oscillations are often linked to changing speed of ER Ca²⁺ efflux, depending on IP₃ receptor activity or IP₃ metabolism (Dupont et al., 2011). In our view, increased IP₃ levels are certainly a trigger of Ca²⁺ oscillations, though it is likely not maintaining the Ca²⁺ oscillation (Figure 2B). ER/cytosol Ca²⁺ signals during spontaneous Ca²⁺ oscillations are in a non-linear (circular) slope relationship with a spatiotemporal time-lag in the range of seconds (Schulte et al., 2022). Furthermore, oscillatory ER Ca²⁺ fluxes can go on for minutes, in presence and absence of extracellular Ca²⁺ (Figure 2B, lower panel) (Schulte et al., 2022).

We would like to suggest the following model: a passive ER Ca²⁺ leak pore, like Sec61a, maybe in concert with other passive ER Ca²⁺ leak mediators (Lemos et al., 2021), mediates a constant ER Ca²⁺ flux to the cytosol. The ER Ca²⁺ leak is powerful and fast enough to shape the spatiotemporal profile of the ER Ca²⁺ oscillations. The Ca²⁺ stays in the cell and the SERCA increases its activity depending on the cytosolic Ca²⁺ concentration. Thus, Ca²⁺-dependency of the SERCA2 (Satoh et al., 2011) might be sufficient to explain the oscillatory behavior of ER-Ca²⁺ influx and efflux (Figure 2B). The weakness of this model is that Sec61a, the Ca²⁺ leak channel that would best fit to the concept, is a highly regulated protein (Daverkausen-Fischer and Pröls, 2022). Still, the sum of all ER leak mechanisms could fulfill the fundamental property of a passive ER Ca²⁺ leak pore through which Ca²⁺ flow is rather fast, for instance as seen after SERCA inhibition. How the ER Ca²⁺ leak mechanisms are regulated, how it’s activity is reduced, enhanced, activated or blocked, remains to be understood.

Mitochondria are also handling Ca²⁺, show mitochondrial Ca²⁺ (_mCa²⁺) oscillations and are involved in the oscillation cycle, and might account for the temporal shift between ER and cytosolic calcium signals (Ishii et al., 2006; Lim et al., 2021). Much focus was put on the function of the mitochondrial Ca²⁺ uniporter complex (MCU complex) (Stefani et al., 2011). However, there is also MCU-independent Ca²⁺ uptake to mitochondria (Garbincius and Elrod, 2022). For instance, in MCU knockout cells, agonist-induced increase in _mCa²⁺ is strongly reduced, or even abolished (Young et al., 2022; Álvarez-Illera et al., 2020). However, _mCa²⁺ oscillations can be MCU-independent, at least in C. elegans (Álvarez-Illera et al., 2020). How mitochondria handle Ca²⁺ during Ca²⁺ oscillations, in response to stimuli or in cases of _mCa²⁺-overload or underload, might be analyzed with triple color imaging experiments. For such experiments, simultaneous Ca²⁺ imaging, e.g. ER Ca²⁺ in green, cytosolic Ca²⁺ in red and mitochondrial Ca²⁺ in far-red, need to be validated.

The ER Ca²⁺-leak shapes agonist-induced Ca²⁺ fluxes

One widely studied Ca²⁺ signal is IP₃-induced Ca²⁺ release (IP₃-iCR). In astrocytes, IP₃-iCR can be activated by adenosine via the highly expressed metabotropic receptor Adora1a (Figure 3A, minimal model). Fast adenosine stimuli activate fast ER Ca²⁺ release and subsequent Ca²⁺ oscillations (Figure 3A, lower panel) (Schulte et al., 2022). ATP, in contrast to adenosine, does not only evoke IP₃-iCR through metabotropic P2Y receptors, but also opens ionotropic P2X receptors (Figure 3B, minimal model). When we stimulated astrocytes with ATP, a long-lasting cytosolic Ca²⁺ signal was induced (Figure 3B, lower panel). This Ca²⁺ entry shoulder did not contribute to ER refilling, but was likely preventing it (Schulte et al., 2022).

FIGURE 3

FIGURE 3. Adenosine- versus ATP-induced Ca²⁺ release in cultured astrocytes. Experiments were performed in presence of extracellular Ca²⁺. (A) Minimal model: Adenosine evokes IP₃-iCR (IP₃, blue). Ca²⁺ oscillations are induced by IP₃-iCR. Ca²⁺ release and Ca²⁺ entry are in balance. Theoretically, Ca²⁺-iCR should contribute to the Ca²⁺ fluxes (indicated in grey). Lower panel: Oscillatory Ca²⁺ cycle induced by adenosine (10 µM) with cytosolic Ca²⁺ signals (magenta) and ER Ca²⁺ signals (green). (B) Minimal model: ATP binds to P2X and P2Y receptors. Metabotropic P2Y receptors evoke IP₃-iCR (blue). P2X receptors might promote depolarizing Ca²⁺ influx, activation of voltage-gated Ca²⁺ channels and ER Ca²⁺ release through Ca²⁺-iCR (cyan). Lower panel: In response to ATP, the cytosolic Ca²⁺ increases while the ER Ca²⁺ decreases. Ca²⁺ entry is induced and creates a signal shoulder in the cytosol. The ER is not refilled during the Ca²⁺ entry shoulder. [Modified according to (Schulte et al., 2022)].

Why is the ATP-induced signal so different from exclusively metabotropic signals? The best explanation is that ATP activates a mixture of IP₃-iCR and Ca²⁺-iCR (Figure 3B). Here, Ca²⁺-iCR would delay the refilling of the ER even though Ca²⁺ entry is ongoing (Figure 3B). In context of above-mentioned data (Figure 2), we think that a pronounced ER Ca²⁺ leak, and not only IP₃-receptors, counteract ER refilling.

The ER Ca²⁺ leak, a fundamental driver of homeostatic Ca²⁺ fluxes

Over the last years, our view on the ER Ca²⁺ leak has drastically changed. The process can no longer be seen as an ‘unavoidable’ side effect of protein translation or as a slow, passive intracellular Ca²⁺ flux. Data from neurons and astrocytes clearly show that resting ER Ca²⁺ leak is upstream of resting Ca²⁺ entry, and thereby indirectly responsible for resting Ca²⁺ levels in the cytosol and ER. The ER Ca²⁺ leak fundamentally shapes cellular Ca²⁺ signals and ER Ca²⁺ oscillations.

One of the most exciting questions for future research is how ER leak channels contribute to microdomain signaling [Ca²⁺-tunnelling (Petersen et al., 2017)], local Ca²⁺ sparks (Cheng and Lederer, 2008), and electrogenic effects for local cellular excitability (Burdakov et al., 2005). Genetically engineered voltage dyes, targeted to the inner-side of the ER-membrane, might help to address electrogenic effects of ER leak channels.

For clinical research, it will be important to know how the ‘Ca²⁺ overload’ phenomenon arises and contributes to mitochondrial dysfunction and cell damage (Mekahli et al., 2011; Garbincius and Elrod, 2022). It can well be that Ca²⁺ overload in the cytosol and in mitochondria is triggered by an increased ER Ca²⁺ leak (ER Ca²⁺ ‘underload’) that continuously promotes resting homeostatic Ca²⁺ influx.

Author contributions

AS and RB conceptualized, wrote, and revised the manuscript. Both authors contributed equally to the article and approved the submitted version.

Funding

The study was funded by the Deutsche Forschungsgemeinschaft (DFG) BL567/3–2, ID Project: 194101929 (RB) and the Evangelisches Studienwerk Villigst (AS).

Acknowledgments

We thank Rohini Gupta for her input.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

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References

Alonso M. T., Rojo-Ruiz J., Navas-Navarro P., Rodriguez-Prados M., Garcia-Sancho J. (2017). Measuring Ca(2+) inside intracellular organelles with luminescent and fluorescent aequorin-based sensors. Biochim. Biophys. Acta. Mol. Cell Res. 1864 (6), 894–899. doi:10.1016/j.bbamcr.2016.12.003

PubMed Abstract | CrossRef Full Text | Google Scholar

Álvarez-Illera P., García-Casas P., Fonteriz R. I., Montero M., Alvarez J. (2020). Mitochondrial Ca(2+) dynamics in MCU knockout C. elegans worms. Int. J. Mol. Sci. 21 (22), E8622. doi:10.3390/ijms21228622

PubMed Abstract | CrossRef Full Text | Google Scholar

Berridge M. J., Bootman M. D., Roderick H. L. (2003). Calcium signalling: Dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4 (7), 517–529. doi:10.1038/nrm1155

PubMed Abstract | CrossRef Full Text | Google Scholar

Birkner A., Konnerth A. (2019). Deep two-photon imaging in vivo with a red-shifted calcium indicator. Methods Mol. Biol. 1929, 15–26. doi:10.1007/978-1-4939-9030-6_2

PubMed Abstract | CrossRef Full Text | Google Scholar

Bootman M. D., Bultynck G. (2020). Fundamentals of cellular calcium signaling: A primer. Cold Spring Harb. Perspect. Biol. 12 (1), a038802. doi:10.1101/cshperspect.a038802

PubMed Abstract | CrossRef Full Text | Google Scholar

Burdakov D., Petersen O. H., Verkhratsky A. (2005). Intraluminal calcium as a primary regulator of endoplasmic reticulum function. Cell calcium 38 (3-4), 303–310. doi:10.1016/j.ceca.2005.06.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Camello C., Lomax R., Petersen O. H., Tepikin A. V. (2002). Calcium leak from intracellular stores--the enigma of calcium signalling. Cell calcium 32 (5-6), 355–361. doi:10.1016/s0143416002001926

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen M., Van Hook M. J., Thoreson W. B. (2015). Ca²⁺ diffusion through endoplasmic reticulum supports elevated intraterminal Ca²⁺ levels needed to sustain synaptic release from rods in darkness. J. Neurosci. 35 (32), 11364–11373. doi:10.1523/JNEUROSCI.0754-15.2015

PubMed Abstract | CrossRef Full Text | Google Scholar

Cheng H., Lederer W. J. (2008). Calcium sparks. Physiol. Rev. 88 (4), 1491–1545. doi:10.1152/physrev.00030.2007

PubMed Abstract | CrossRef Full Text | Google Scholar

Daverkausen-Fischer L., Pröls F. (2022). Regulation of calcium homeostasis and flux between the endoplasmic reticulum and the cytosol. J. Biol. Chem. 298, 102061. doi:10.1016/j.jbc.2022.102061

PubMed Abstract | CrossRef Full Text | Google Scholar

de Juan-Sanz J., Holt G. T., Schreiter E. R., de Juan F., Kim D. S., Ryan T. A. (2017). Axonal endoplasmic reticulum Ca²⁺ content controls release probability in CNS nerve terminals. Neuron 93 (4), 867–881. doi:10.1016/j.neuron.2017.01.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Dupont G., Combettes L., Bird G. S., Putney J. W. (2011). Calcium oscillations. Cold Spring Harb. Perspect. Biol. 3 (3), a004226. doi:10.1101/cshperspect.a004226

PubMed Abstract | CrossRef Full Text | Google Scholar

Flourakis M., Van Coppenolle F., Lehen'kyi V., Beck B., Skryma R., Prevarskaya N. (2006). Passive calcium leak via translocon is a first step for iPLA2-pathway regulated store operated channels activation. FASEB J. official Publ. Fed. Am. Soc. Exp. Biol. 20 (8), 1215–1217. doi:10.1096/fj.05-5254fje

PubMed Abstract | CrossRef Full Text | Google Scholar

Garbincius J. F., Elrod J. W. (2022). Mitochondrial calcium exchange in physiology and disease. Physiol. Rev. 102 (2), 893–992. doi:10.1152/physrev.00041.2020

PubMed Abstract | CrossRef Full Text | Google Scholar

Hartmann J., Karl R. M., Alexander R. P., Adelsberger H., Brill M. S., Ruhlmann C., et al. (2014). STIM1 controls neuronal Ca²⁺ signaling, mGluR1-dependent synaptic transmission, and cerebellar motor behavior. Neuron 82 (3), 635–644. doi:10.1016/j.neuron.2014.03.027

PubMed Abstract | CrossRef Full Text | Google Scholar

Hasel P., Dando O., Jiwaji Z., Baxter P., Todd A. C., Heron S., et al. (2017). Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism. Nat. Commun. 8, 15132. doi:10.1038/ncomms15132

PubMed Abstract | CrossRef Full Text | Google Scholar

Hofer A. M., Machen T. E. (1993). Technique for in situ measurement of calcium in intracellular inositol 1, 4, 5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2. Proc. Natl. Acad. Sci. U. S. A. 90 (7), 2598–2602. doi:10.1073/pnas.90.7.2598

PubMed Abstract | CrossRef Full Text | Google Scholar

Kipanyula M. J., Contreras L., Zampese E., Lazzari C., Wong A. K., Pizzo P., et al. (2012). Ca²⁺ dysregulation in neurons from transgenic mice expressing mutant presenilin 2. Aging Cell 11 (5), 885–893. doi:10.1111/j.1474-9726.2012.00858.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Ishii Kiyoaki, Hirose Kenzo, Iino Masamitsu (2006). Ca²⁺ shuttling between endoplasmic reticulum and mitochondria underlying Ca²⁺ oscillations. EMBO Rep. 7 (4), 390–396. doi:10.1038/sj.embor.7400620

PubMed Abstract | CrossRef Full Text | Google Scholar

Lalonde J., Saia G., Gill G. (2014). Store-operated calcium entry promotes the degradation of the transcription factor Sp4 in resting neurons. Sci. Signal. 7 (328), ra51. doi:10.1126/scisignal.2005242

PubMed Abstract | CrossRef Full Text | Google Scholar

Lang S., Pfeffer S., Lee P. H., Cavalie A., Helms V., Forster F., et al. (2017). An update on Sec61 channel functions, mechanisms, and related diseases. Front. Physiol. 8, 887. doi:10.3389/fphys.2017.00887

PubMed Abstract | CrossRef Full Text | Google Scholar

Laude A. J., Tovey S. C., Dedos S. G., Potter B. V., Lummis S. C., Taylor C. W. (2005). Rapid functional assays of recombinant IP3 receptors. Cell calcium 38 (1), 45–51. doi:10.1016/j.ceca.2005.04.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Lemos F. O., Bultynck G., Parys J. B. (2021). A comprehensive overview of the complex world of the endo- and sarcoplasmic reticulum Ca(2+)-leak channels. Biochim. Biophys. Acta. Mol. Cell Res. 1868 (7), 119020. doi:10.1016/j.bbamcr.2021.119020

PubMed Abstract | CrossRef Full Text | Google Scholar

Lim D., Semyanov A., Genazzani A., Verkhratsky A. (2021). Calcium signaling in neuroglia. Int. Rev. Cell Mol. Biol. 362, 1–53. doi:10.1016/bs.ircmb.2021.01.003

PubMed Abstract | CrossRef Full Text | Google Scholar

Mekahli D., Bultynck G., Parys J. B., De Smedt H., Missiaen L. (2011). Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb. Perspect. Biol. 3 (6), a004317. doi:10.1101/cshperspect.a004317

PubMed Abstract | CrossRef Full Text | Google Scholar

Petersen O. H., Courjaret R., Machaca K. (2017). Ca(2+) tunnelling through the ER lumen as a mechanism for delivering Ca(2+) entering via store-operated Ca(2+) channels to specific target sites. J. Physiol. 595 (10), 2999–3014. doi:10.1113/JP272772

PubMed Abstract | CrossRef Full Text | Google Scholar

Prada J., Sasi M., Martin C., Jablonka S., Dandekar T., Blum R. (2018). An open source tool for automatic spatiotemporal assessment of calcium transients and local 'signal-close-to-noise' activity in calcium imaging data. PLoS Comput. Biol. 14 (3), e1006054. doi:10.1371/journal.pcbi.1006054

PubMed Abstract | CrossRef Full Text | Google Scholar

Putney J. W., Steinckwich-Besancon N., Numaga-Tomita T., Davis F. M., Desai P. N., D'Agostin D. M., et al. (2017). The functions of store-operated calcium channels. Biochim. Biophys. Acta. Mol. Cell Res. 1864 (6), 900–906. doi:10.1016/j.bbamcr.2016.11.028

PubMed Abstract | CrossRef Full Text | Google Scholar

Rehberg M., Lepier A., Solchenberger B., Osten P., Blum R. (2008). A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum. Cell calcium 44 (4), 386–399. doi:10.1016/j.ceca.2008.02.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Rodriguez-Garcia A., Rojo-Ruiz J., Navas-Navarro P., Aulestia F. J., Gallego-Sandin S., Garcia-Sancho J., et al. (2014). GAP, an aequorin-based fluorescent indicator for imaging Ca²⁺ in organelles. Proc. Natl. Acad. Sci. U. S. A. 111 (7), 2584–2589. doi:10.1073/pnas.1316539111

PubMed Abstract | CrossRef Full Text | Google Scholar

Rodríguez-Prados M., Rojo-Ruiz J., García-Sancho J., Alonso M. T. (2020). Direct monitoring of ER Ca²⁺ dynamics reveals that Ca²⁺ entry induces ER-Ca²⁺ release in astrocytes. Pflugers Arch. 472 (4), 439–448. doi:10.1007/s00424-020-02364-7

PubMed Abstract | CrossRef Full Text | Google Scholar

Rodriguez-Prados M., Rojo-Ruiz J., Garcia-Sancho J., Alonso M. T. (2020). Direct monitoring of ER Ca(2+) dynamics reveals that Ca(2+) entry induces ER-Ca(2+) release in astrocytes. Pflugers Arch. 472 (4), 439–448. doi:10.1007/s00424-020-02364-7

PubMed Abstract | CrossRef Full Text | Google Scholar

Rossi A. M., Taylor C. W. (2020). Reliable measurement of free Ca(2+) concentrations in the ER lumen using Mag-Fluo-4. Cell calcium 87, 102188. doi:10.1016/j.ceca.2020.102188

PubMed Abstract | CrossRef Full Text | Google Scholar

Stefani Diego De, Raffaello Anna, Teardo Enrico, Szabò Ildikò, Rizzuto Rosario (2011). A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476 (7360), 336–340. doi:10.1038/nature10230

PubMed Abstract | CrossRef Full Text | Google Scholar

Samtleben S., Jaepel J., Fecher C., Andreska T., Rehberg M., Blum R. (2013). Direct imaging of ER calcium with targeted-esterase induced dye loading (TED). J. Vis. Exp. 75 (75), e50317. doi:10.3791/50317

PubMed Abstract | CrossRef Full Text | Google Scholar

Samtleben S., Wachter B., Blum R. (2015). Store-operated calcium entry compensates fast ER calcium loss in resting hippocampal neurons. Cell calcium 58 (2), 147–159. doi:10.1016/j.ceca.2015.04.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Satoh K., Matsu-ura T., Enomoto M., Nakamura H., Michikawa T., Mikoshiba K. (2011). Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase (SERCA) 2a pump activity on cytosolic calcium in living cells. J. Biol. Chem. 286 (23), 20591–20599. doi:10.1074/jbc.M110.204685

PubMed Abstract | CrossRef Full Text | Google Scholar

Schäuble N., Lang S., Jung M., Cappel S., Schorr S., Ulucan O., et al. (2012). BiP-mediated closing of the Sec61 channel limits Ca²⁺ leakage from the ER. EMBO J. 31 (15), 3282–3296. doi:10.1038/emboj.2012.189

PubMed Abstract | CrossRef Full Text | Google Scholar

Schulte A., Bieniussa L., Gupta R., Samtleben S., Bischler T., Doering K., et al. (2022). Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit. Cell calcium 101, 102515. doi:10.1016/j.ceca.2021.102515

PubMed Abstract | CrossRef Full Text | Google Scholar

Solovyova N., Verkhratsky A. (2002). Monitoring of free calcium in the neuronal endoplasmic reticulum: An overview of modern approaches. J. Neurosci. Methods 122 (1), 1–12. doi:10.1016/s0165-0270(02)00300-x

PubMed Abstract | CrossRef Full Text | Google Scholar

Solovyova N., Veselovsky N., Toescu E. C., Verkhratsky A. (2002). Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: Direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry. EMBO J. 21 (4), 622–630. doi:10.1093/emboj/21.4.622

PubMed Abstract | CrossRef Full Text | Google Scholar

Streb H., Irvine R. F., Berridge M. J., Schulz I. (1983). Release of Ca²⁺ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1, 4, 5-trisphosphate. Nature 306 (5938), 67–69. doi:10.1038/306067a0

PubMed Abstract | CrossRef Full Text | Google Scholar

Suzuki J., Kanemaru K., Ishii K., Ohkura M., Okubo Y., Iino M. (2014). Imaging intraorganellar Ca²⁺ at subcellular resolution using CEPIA. Nat. Commun. 5, 4153. doi:10.1038/ncomms5153

PubMed Abstract | CrossRef Full Text | Google Scholar

Tang S., Wong H-C., Wang Z-M., Huang Y., Zou J., Zhuo Y., et al. (2011). Design and application of a class of sensors to monitor Ca²⁺ dynamics in high Ca²⁺ concentration cellular compartments. Proc. Natl. Acad. Sci. U. S. A. 108 (39), 16265–16270. doi:10.1073/pnas.1103015108

PubMed Abstract | CrossRef Full Text | Google Scholar

Thastrup O., Dawson A. P., Scharff O., Foder B., Cullen P. J., Drobak B. K., et al. (1989). Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 27 (1-2), 17–23. doi:10.1007/BF02222186

PubMed Abstract | CrossRef Full Text | Google Scholar

Verkhratsky A., Nedergaard M. (2018). Physiology of astroglia. Physiol. Rev. 98 (1), 239–389. doi:10.1152/physrev.00042.2016

PubMed Abstract | CrossRef Full Text | Google Scholar

Verkhratsky A. (2005). Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol. Rev. 85 (1), 201–279. doi:10.1152/physrev.00004.2004

PubMed Abstract | CrossRef Full Text | Google Scholar

Young Michael, Schug Zachary T., Booth David, Yule David, Mikoshiba Katsuhiko, Hajnoczky Gyorgy, et al. (2022). Metabolic adaptation to the chronic loss of Ca(2+) signaling induced by KO of IP3 receptors or the mitochondrial Ca(2+) uniporter. J. Biol. Chem. 298 (1), 101436. doi:10.1016/j.jbc.2021.101436

PubMed Abstract | CrossRef Full Text | Google Scholar