Caffeine chelates calcium in the lumen of the endoplasmic reticulum

Cytosolic Ca²⁺ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca²⁺ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca²⁺ levels. Caffeine sensitizes RyR to Ca²⁺ and promotes ER Ca²⁺ release at basal cytosolic Ca²⁺ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca²⁺ with the low-affinity genetic Ca²⁺ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca²⁺ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca²⁺ release does not go along with the expected cytosolic Ca²⁺ increase. These results can be explained by Ca²⁺ chelation by caffeine inside the ER. Ca²⁺-overloaded mitochondria also display a drop of the matrix Ca²⁺ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca²⁺ concentration (10^-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca²⁺ release) and in the cytosol (increase in Ca²⁺ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca²⁺ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.