Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca 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 Ca channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca levels. Caffeine sensitizes RyR to Ca and promotes ER Ca release at basal cytosolic Ca levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca with the low-affinity genetic Ca probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca release does not go along with the expected cytosolic Ca increase. These results can be explained by Ca chelation by caffeine inside the ER. Ca-overloaded mitochondria also display a drop of the matrix Ca concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca concentration (10 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca release) and in the cytosol (increase in Ca 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 Ca 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.