MICU1 and MICU2 potentiation of Ca(2+) uptake by the mitochondrial Ca(2+) uniporter of Trypanosoma cruzi and its inhibition by Mg(2).

The mitochondrial Ca(2+) uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca(2+) 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 Ca(2+) overload at low cytosolic Ca(2+) levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca(2+) uptake at low extramitochondrial Ca(2+) concentrations ([Ca(2+)](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 Ca(2+) uptake at low [Ca(2+)](ext), significantly decreased the mitochondrial Ca(2+) uptake at different [Ca(2+)](ext), without dissipation of the mitochondrial membrane potential, and increased the [Ca(2+)](ext) set point needed for Ca(2+) uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg(2+) was found to be a negative regulator of MCUC-mediated mitochondrial Ca(2+) uptake at different [Ca(2+)](ext). Occlusion of the MCUC pore by Mg(2+) could partially explain the lack of mitochondrial Ca(2+) uptake at low [Ca(2+)](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.