MICU1 and MICU2 potentiation of Ca2+ uptake by the mitochondrial Ca2+ uniporter of Trypanosoma cruzi and its inhibition by Mg2.

The mitochondrial Ca2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca2+ signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca2+ overload at low cytosolic Ca2+ levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca2+ uptake at low extramitochondrial Ca2+ concentrations ([Ca2+]ext) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca2+ uptake at low [Ca2+]ext, significantly decreased the mitochondrial Ca2+ uptake at different [Ca2+]ext, without dissipation of the mitochondrial membrane potential, and increased the [Ca2+]ext set point needed for Ca2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca2+ uptake at different [Ca2+]ext. Occlusion of the MCUC pore by Mg2+ could partially explain the lack of mitochondrial Ca2+ uptake at low [Ca2+]ext in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.