Shaped by leaky ER: Homeostatic Ca2+ fluxes

Abstract

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.

Keywords: Ca2+ homeostasis; Ca2+ ion analysis; Ca2+ leak; Ca2+ oscillation; ER Ca2+ imaging; ER Ca2+ store; SERCA; store-operated Ca2+ entry.

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)].

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)].

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)].