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The mitochondrial calcium uniporter is a Ca-activated Ca channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca-binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca signals.