The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca2+ entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca2+ uniporter. To clarify the role of UCPs in mitochondrial Ca2+ handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca2+ and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca2+ uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca2+ elevations evoked by histamine. The reduced Ca2+ elevations were due to inhibition of store-operated Ca2+ entry and reduced depletion of endoplasmic reticulum (ER) Ca2+ stores. UCP3 depletion accelerated the ER Ca2+ refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca2+ (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca2+ responses. Our results indicate that UCP3 is not a mitochondrial Ca2+ uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca2+ thus reflect metabolic alterations that impact on cellular Ca2+ homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca2+ uptake and release sites

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