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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.

Mitochondria Take Up Ca<sup>2+</sup> in Two Steps Dependently on Store-Operated Ca<sup>2+</sup> Entry in Mast Cells