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.

Mayara S Bertolini, Roberto Docampo. Cell Calcium. 2022 Sep 21;107:102654. doi: 10.1016/j.ceca.2022.102654.