FIGURE 5.

Effect of SERCA inhibition or cell permeabilization on mitochondrial Ca²⁺ elevation. A, the same protocols and conditions as in Fig. 2 were used to deplete Ca²⁺ stores and to monitor Ca²⁺ release and then the influx component in mitochondria, but in the presence of the SERCA pump inhibitor 1 μm TG. Histamine was 100 μm. Left, average of [Ca²⁺]_mit recordings from Ctrl (scramble) siRNA or the UCP3 siRNA cells. Right, statistical evaluation of the UCP3 knockdown on the integrated [Ca²⁺]_mit responses evoked by histamine or after Ca²⁺ readmission, from data shown in the left panel. Bars are mean ± S.E. of 51 (n = 5) and 45 cells (n = 4) for Ctrl and UCP3 siRNA, respectively. NS, not stimulated. AUC, area under the curve. B, Ru360-sensitive mitochondrial Ca²⁺-uptake in permeabilized cells, in ATP-depleted medium. HeLa cells were transiently co-transfected with the mitochondrial calcium probe 4mtD3cpv and the indicated siRNAs. After permeabilization with digitonin, [Ca²⁺]_mit was measured in intracellular buffer. Left, original [Ca²⁺]_mit recordings of permeabilized HeLa cells during the addition of 3.5 μm free Ca²⁺ in the presence of or after wash-out of the mitochondrial Ca²⁺ uniporter inhibitor Ru360 (10 μm). Right, statistical evaluation of UCP3 siRNA effects on the slope of Ca²⁺ elevation. For the latter statistics, the Ca²⁺ responses for each trace were fitted with a linear function, and the Ru360-dependent slope was subtracted from the following one in the absence of the inhibitor. Bars are mean ± S.E. of 79 (n = 6) and 67 (n = 6) cells for Ctrl and UCP3 siRNA, respectively.