Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells

MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter protein, mitochondrial) that has been shown recently to act as a gatekeeper of the MCU at low [Ca2+](c) (cytosolic [Ca2+]). In the present study we have investigated in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we have found several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+ uptake was largely increased at a low [Ca2+](c) (<2 mu M), but it was decreased at a high [Ca2+](c) (>4 mu M). In the 2-4 mu M range a mixed behaviour was observed, where mitochondrial Ca2+ uptake started earlier in the MICU1-silenced cells, but at a lower rate than in the controls. The sensitivity of Ca2+ uptake to Ruthenium Red and Ru360 was similar at both high and low [Ca2+](c), indicating that the same Ca2+ pathway was operating in both cases. The increased Ca2+-uptake rate observed at a [Ca2+](c) below 2 mu M was transient and became inhibited during Ca2+ entry. Development of this inhibition was slow, requiring 5 min for completion, and was hardly reversible. Therefore MICU1 acts both as a MCU gatekeeper at low [Ca2+](c) and as a cofactor necessary to reach the maximum Ca2+-uptake rate at high [Ca2+](c). Moreover, in the absence of MICU1, the MCU becomes sensitive to a slow-developing inhibition that requires prolonged increases in [Ca2+](c) in the low micromolar range.