Mitochondrial Ca²⁺ influx mechanisms during cytosolic/mitochondrial Ca²⁺ transient. The top of the figure is an example of Ca²⁺ transient at microdomains between mitochondria and ER/SR ([Ca²⁺]_ER-mito; red line) and Ca²⁺ transient at the mitochondrial matrix ([Ca²⁺]_m; blue line). RaM (black) shows 50-fold faster Ca²⁺ transport compared with the MCU, and the activation peak is at 50 nM of extra-mitochondrial Ca²⁺. Letm1 (orange) can be activated at ≥200 nM of extra-mitochondrial Ca²⁺, but at high [Ca²⁺]_ER-mito condition, a role of Letm1 shifts to Ca²⁺ efflux rather than Ca²⁺ uptake into the mitochondrial matrix. mRyR1 (red) can start to be activated at 1 µM of extra-mitochondrial Ca²⁺, with a fivefold faster Ca²⁺ transport compared with the MCU. 2 µM is the half-maximal concentration for Ca²⁺-dependent activation of mRyR1, and 20 µM is the half-maximal concentration for Ca²⁺-dependent inhibition. Thus, mRyR1 inactivates before [Ca²⁺]_ER-mito reaches the peak. A lower concentration of extra-mitochondrial Ca²⁺ (such as 200–300 nM) does not activate MCU, and at least >1 µM Ca²⁺ is needed for the initial activation. The estimated half-maximal concentration for the activation of MCU is ≅20 mM.