Mitochondrial Ca²⁺ concentrations in live cells: quantification methods and discrepancies

Intracellular Ca 2+ signaling controls numerous cellular functions. Mitochondria respond to cytosolic Ca 2+ changes by adapting mitochondrial functions and, in some cell types, shaping the spatiotemporal properties of the cytosolic Ca 2+ signal. Numerous methods have been developed to specifically and quantitatively measure the mitochondrial‐free Ca 2+ concentrations ([Ca 2+ ] m ), but there are still significant discrepancies in the calculated absolute values of [Ca 2+ ] m in stimulated live cells. These discrepancies may be due to the distinct properties of the methods used to measure [Ca 2+ ] m , the calcium‐free/bound ratio, and the cell‐type and stimulus‐dependent Ca 2+ dynamics. Critical processes happening in the mitochondria, such as ATP generation, ROS homeostasis, and mitochondrial permeability transition opening, depend directly on the [Ca 2+ ] m values. Thus, precise determination of absolute [Ca 2+ ] m values is imperative for understanding Ca 2+ signaling. This review summarizes the reported calibrated [Ca 2+ ] m values in many cell types and discusses the discrepancies among these values. Areas for future research are also proposed.