Mitochondria Take Up Ca²⁺ in Two Steps Dependently on Store-Operated Ca²⁺ Entry in Mast Cells

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The ability of mitochondria to take up Ca²⁺ has important functional implications for modulation of cellular Ca²⁺ signaling. Mitochondrial Ca²⁺ uptake is stimulated by an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Here, we found that the increase in mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) occurs in two steps in a single antigen-activated mast cell in the presence of extracellular Ca²⁺ (1.0 mM). The two-step elevation of [Ca²⁺]_m was also observed after adding thapsigargin, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase. The proportion of mitochondria showing the two-step Ca²⁺ elevation dropped off in direct accord with decrease in extracellular Ca²⁺ concentration. The second step of the [Ca²⁺]_m increase was suppressed significantly in the absence of extracellular Ca²⁺ and in knockdown cells of stromal interaction molecule 1 (STIM1), an essential molecule on endoplasmic reticulum (ER) membrane for store-operated Ca²⁺ entry, in the presence of extracellular Ca²⁺ (1.0 mM), while the first elevation was not affected in either case. The results indicate that mitochondria take up cytosolic Ca²⁺ in two steps; first and second uptakes are derived from the Ca²⁺ release from ER and the Ca²⁺ influx through store-operated Ca²⁺ channels, respectively. Additionally, rotenone and antimycin A, which are inhibitors of mitochondrial electron transport complex I and III, respectively, diminished mitochondrial Ca²⁺ uptake and significantly suppressed degranulation stimulated with antigen. The mitochondrial Ca²⁺ uptake may modulate mast cell function by regulating the [Ca²⁺]_c.