MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions

Abstract

Mitochondrial Ca 2+  uptake is mediated by the mitochondrial uniporter complex (mtCU) that includes a tetramer of the pore-forming subunit, MCU, a scaffold protein, EMRE, and the EF-hand regulatory subunit, MICU1 either homodimerized or heterodimerized with MICU2/3. MICU1 has been proposed to regulate Ca 2+ uptake via the mtCU by physically occluding the pore and preventing Ca 2+ flux at resting cytoplasmic [Ca 2+ ] (free calcium concentration) and to increase Ca 2+ flux at high [Ca 2+ ] due to cooperative activation of MICUs EF-hands. However, mtCU and MICU1 functioning when its EF-hands are unoccupied by Ca 2+ is poorly studied due to technical limitations. To overcome this barrier, we have studied the mtCU in divalent-free conditions by assessing the Ru265-sensitive Na +  influx using fluorescence-based measurement of mitochondrial matrix [Na + ] (free sodium concentration) rise and the ensuing depolarization and swelling. We show an increase in all these measures of Na + uptake in MICU1KO cells as compared to wild-type (WT) and rescued MICU1KO HEK cells. However, mitochondria in WT cells and MICU1 stable-rescued cells still allowed some Ru265-sensitive Na + influx that was prevented by MICU1 in excess upon acute overexpression. Thus, MICU1 restricts the cation flux across the mtCU in the absence of Ca 2+ , but even in cells with high endogenous MICU1 expression such as HEK, some mtCU seem to lack MICU1-dependent gating. We also show rearrangement of the mtCU and altered number of functional channels in MICU1KO and different rescues, and loss of MICU1 during mitoplast preparation, that together might have obscured the pore-blocking function of MICU1 in divalent-free conditions in previous studies.