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

摘要

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.