Ca2+ Hot Spots on the Mitochondrial Surface Are Generated by Ca2+ Mobilization from Stores, but Not by Activation of Store-Operated Ca2+ Channels

Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca2+ during stimulation because of their vicinity to microdomains of high [Ca2+], the direct proof of Ca2+ hot spots' existence is still lacking. Thanks to a GFP-based Ca2+ probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca2+ mobilization, the [Ca2+] in small regions of the mitochondrial surface reaches levels 5- to 10-fold higher than in the bulk cytosol. We also show that the [Ca2+] to which mitochondria are exposed during capacitative Ca2+ influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2- to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca2+ channels. These results demonstrate that mitochondria are exposed to Ca2+ hot spots close to the ER but are excluded from the regions where capacitative Ca2+ influx occurs.