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

The mitochondrial Ca uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca 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 overload at low cytosolic Ca 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 uptake at low extramitochondrial Ca concentrations ([Ca]) 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 uptake at low [Ca], significantly decreased the mitochondrial Ca uptake at different [Ca], without dissipation of the mitochondrial membrane potential, and increased the [Ca] set point needed for Ca uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg was found to be a negative regulator of MCUC-mediated mitochondrial Ca uptake at different [Ca]. Occlusion of the MCUC pore by Mg could partially explain the lack of mitochondrial Ca uptake at low [Ca] 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.