Leucine Zipper EF Hand-containing Transmembrane Protein 1 (Letm1) and Uncoupling Proteins 2 and 3 (UCP2/3) Contribute to Two Distinct Mitochondrial Ca2+ Uptake Pathways

Cytosolic Ca2+ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca2+ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca2+/H+ antiporter that achieved mitochondrial Ca2+ sequestration at small Ca2+ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca2+ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca2+ but strongly diminished the transfer of entering Ca2+ into mitochondria, subsequently, resulting in a reduction of store-operated Ca2+ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca2+ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca2+ uptake at low Ca2+ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca2+ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca2+ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca2+ uptake pathways in intact endothelial cells.