Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane

Abstract

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca2+ concentration: an initial transient release of Ca2+ from intracellular stores is followed by a sustained phase of Ca2+ influx. This influx is generally store-dependent and is required for controlling a host of Ca2+-dependent processes ranging from exocytosis to cell growth and proliferation. In many cell types, store-operated Ca2+ entry is manifest as a non-voltage-gated Ca2+ current called ICRAC (Ca2+ release-activated Ca2+ current). Just how store emptying activates CRAC channels remains unclear, and some of our recent experiments that address this issue will be described. No less important from a physiological perspective is the weak Ca2+ buffer paradox: whereas macroscopic (whole cell) ICRAC can be measured routinely in the presence of strong intracellular Ca2+ buffer, the current is generally not detectable under physiological conditions of weak buffering following store emptying with the second messenger InsP3. In this review, I describe some of our experiments aimed at understanding just why InsP3 is ineffective under these conditions and which lead us to conclude that respiring mitochondria are essential for the activation of ICRAC in weak intracellular Ca2+ buffer. Mitochondrial Ca2+ uptake also increases the dynamic range over which InsP3 functions as the second messenger that controls Ca2+ influx. Finally, we find that Ca2+-dependent slow inactivation of Ca2+ influx, a widespread but poorly understood phenomenon that helps shape the profile of an intracellular Ca2+ signal, is regulated by mitochondrial Ca2+ buffering. Thus, by enabling macroscopic store-operated Ca2+ current to activate and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca2+ influx. Store-operated Ca2+ entry reflects therefore a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

Figure 1. Development of I_CRAC following store depletion

A, the upper panel shows the typical voltage ramp protocol used to monitor I_CRAC in mast cells, the related rat basophilic leukaemia (RBL-1) cell line and jurkat T-lymphocytes. The ramp spans −100 to +100 mV in 50 ms, and is applied from a holding potential of 0 mV once every 2 s. The lower panel depicts the time course of activation of I_CRAC. The current amplitude, measured at −80 mV from each ramp, has been normalised for cell size by dividing the current by cell capacitance. The thick line shows a mono-exponential fit to the activation time course. B shows the current-voltage relationship, once the current had reached steady state. Note the inward rectification and very positive reversal potential, characteristic of I_CRAC. The pipette solution is caesium glutamate based, supplemented with InsP₃ and 10 mm EGTA.

Figure 2. The three contemporary models proposed to account for the activation mechanism of I_CRAC

Figure 3. The weak buffer paradox

A, InsP₃ generally fails to activate I_CRAC in weak intracellular Ca²⁺ buffer (0.1 mm EGTA) whereas it is effective in strong buffer (10 mm EGTA). B, current-voltage relationships for the two recordings from A, taken at steady state. C, the mean amplitude of I_CRAC (left-hand panel) and the fraction of cells responding (right-hand panel) for each condition is shown. In this and all subsequent figures, data from RBL-1 cells are shown. ^*P < 0.01.

Figure 4. InsP₃ still fails to activate I_CRAC in weak Ca²⁺ buffer at physiological temperature

Experiments were carried out at 36 °C. A, whereas InsP₃ evoked a large I_CRAC at 36 °C in 10 mm EGTA, no current was detectable in 0.1 mm EGTA. B, aggregate data are summarised. ^*P < 0.01 (Student's t test).

Figure 5. Passive activation of I_CRAC

A, comparison of the time course of development of I_CRAC following dialysis with either 10 mm EGTA alone or 30 μm InsP₃+ EGTA. Note the initial slow development of I_CRAC followed by a second, faster phase in response to EGTA alone. B, development of I_CRAC following dialysis with 10 mm BAPTA or dimethyl BAPTA (panel C).

Figure 6. SERCA pumps shape the pattern of passive activation of I_CRAC

A, inclusion of cyclopiazonic acid (CPA) eliminates the initial slow phase of current development, leaving the second component intact. B, following dialysis with 2.5 mm EGTA, I_CRAC activated partially in this cell. Subsequent application of thapsigargin increased the extent of the current, as well as its rate of development.

Figure 7. SERCA pumps function in the presence of InsP₃

A, the relationship between intrapipette EGTA concentration and amplitude of I_CRAC (evoked by dialysis with 30 μm InsP₃) is plotted. Note that I_CRAC was clearly submaximal in the presence of 0.6 mm EGTA. Each point is the mean ±s.e.m. of at least 5 cells. B, recordings are shown for a cell dialysed with 30 μm InsP₃ and 0.6 mm EGTA (•) and for one dialysed with this solution but supplemented with 2 μm thapsigargin, a SERCA blocker (○). Note the increase in the size of I_CRAC following inhibition of the pumps. C, the amplitudes, delays and times to peak are summarised for experiments as in B. Filled circles denote InsP₃+ 0.6 mm EGTA whereas open cirlces represent InsP₃+ 0.6 mm EGTA + thapsigargin.

Figure 8. InsP₃ activates I_CRAC in weak buffer provided SERCA pump activity is compromised

A, InsP₃ together with thapsigargin activates I_CRAC in weak buffer whereas InsP₃ alone is ineffective. B, InsP₃ with cyclopiazonic acid, a structurally distinct SERCA pump blocker, is also effective. SERCA pump block alone (2 μm Thap + 0.1 mm EGTA) can also activate I_CRAC, albeit at a much slower rate.

Figure 9. SERCA pump block is sufficient to activate I_CRAC even in high intracellular Ca²⁺

A, InsP₃ and thapsigargin can activate I_CRAC in weak buffer even in the presence of high intracellular Ca²⁺ (100 μm total CaCl₂ in the pipette solution, free Ca²⁺ concentration estimated to be in the micromolar range). Thapsigargin alone was also able to evoke I_CRAC under these conditions, but after a delay and at a slower rate. B, aggregate data are summarised.

Figure 10. The combination of InsP₃ and thapsigargin evokes a robust secretory response in weak buffer, whereas InsP₃ alone is largely ineffective

InsP₃ alone (•) or in combination with GTPγS (▵) did not trigger a detectable increase in membrane capacitance, whereas InsP₃ together with thapsigargin and GTPγS produced a prominent secretory response (○). In these experiments, cells were clamped at −60 mV.

Figure 11. InsP₃ activates I_CRAC in weak buffer provided mitochondria are energised

A, time course of activation of I_CRAC in the presence of the mitochondrial cocktail (○). I_CRAC does not activate in weak Ca²⁺ buffer in response to InsP₃ alone (•). B, steady-state I-V relationships are shown for the cells in A. C, the effects of cocktail are suppressed by preventing mitochondrial Ca²⁺ uptake by either depolarising mitochondria (pre-treatment with antimycin A (5 μg ml⁻¹) and oligomycin (0.5 μg ml⁻¹)) or inhibiting the uniporter with ruthenium red. D, the presence of cocktail dramatically increases the fraction of cells that respond to InsP₃ in weak Ca²⁺ buffer. ^*P < 0.01.

Figure 12. Physiological antagonism between SERCA pumps and mitochondria

A, the potentiation of I_CRAC by cocktail is suppressed by strong intracellular Ca²⁺ buffer (10 mm EGTA), indicating that the effect is Ca²⁺-dependent. B, the extent of I_CRAC to InsP₃ in weak buffer alone, in mitochondrial cocktail and cocktail plus thapsigargin is compared. ^*P < 0.01.

Figure 13. Energised mitochondria increase the ability of InsP₃ to activate I_CRAC in weak Ca²⁺ buffer

A, whereas 5 μm InsP₃ is ineffective in weak buffer (○), it evokes a prominent I_CRAC in the presence of energised mitochondria (•). B, I-V relationships for the cells shown in A, taken at steady state. C, concentration-response curve to InsP₃ in the absence (○) and presence (•) of cocktail. D, the fraction of cells responding versus concentration of InsP₃ is plotted in the absence (○) and presence (•) of energised mitochondria.

Figure 14. Mitochondrial Ca²⁺ uptake reduces the rate and extent of Ca²⁺-dependent slow inactivation

A, dialysis with InsP₃ and thapsigargin in weak buffer (control) activates I_CRAC but then the current inactivates slowly (•). The extent of inactivation is reduced if mitochondria are energised (○). B and C summarise aggregate data comparing the extent (B) and rate (C) of inactivation of I_CRAC in control cells, in those in which cocktail was added to the solution and in cells exposed to cocktail and ruthenium red, to inhibit mitochondrial Ca²⁺ uptake. ^*P < 0.01. t_1/2, time at which I_CRAC had inactivated by 50 %.

Figure 15. A diagrammatic scheme for the role of mitochondria in controlling I_CRAC under physiological conditions of weak intracellular Ca²⁺ buffering

A, the resting state, where I_CRAC is not functioning, is shown. Stores are largely full and any Ca²⁺ that leaks from the stores is taken back up by the SERCA pumps. B, increasing InsP₃ levels in the absence of active mitochondrial Ca²⁺ uptake, releases Ca²⁺ from the stores. However, the SERCA pumps are able to resequester sufficient Ca²⁺ so only a very small fraction of CRAC channels are activated (undetectable in whole cell mode). Furthermore, the rise in cytosolic Ca²⁺ results in Ca²⁺-dependent slow inactivation of CRAC channels, and possibly InsP₃ receptors as well. C, in the presence of respiring mitochondria, InsP₃ activates macroscopic I_CRAC. Ca²⁺ released from the stores by InsP₃ is taken up by mitochondria. This reduces the amount of Ca²⁺ available to the SERCA pumps and in the vicinity of open InsP₃ receptors such that the stores are depleted sufficiently for macroscopic I_CRAC to activate (less refilling by SERCA pumps and less inactivation of InsP₃ receptors). Some refilling does occur because inclusion of thapsigargin enhances the size of the current. Furthermore, mitochondrial Ca²⁺ buffering reduces the rate and extent of Ca²⁺-dependent slow inactivation, thereby increasing the size and duration of the current. D, a simplified gating scheme for CRAC channels summarising the role of mitochondrial Ca²⁺ buffering. Mitochondria facilitate opening (Closed to Open transition) whilst, simultaneously, reducing inactivation (Open to Inactivated transition). In this way, mitochondria have a much larger impact on I_CRAC than through either transition alone.