Mitochondrial Calcium and Bioenergetics Controlled by Tight Coordination of MCU and NCLX

Abstract

Mitochondrial Ca2+ is regulated by Ca2+ transport proteins in the mitochondrial inner membrane. Among these are the mitochondrial calcium uniporter (MCU), a Ca2+ channel responsible for most Ca2+ uptake in mitochondria, and NCLX, a Na+/Ca2+ exchanger, mediating Ca2+ release to the cytosol. After more than five decades of intensive study, the MCU gene was finally cloned in 2010. Strikingly, MCU-KO mouse, whose mitochondria lose >90% Ca2+ uptake capacity, was born normally with very mild phenotype and only slight reduction of mitochondrial Ca2+. How does the animal maintain normal physiology without MCU, which is highly conserved and tightly regulated in most eukaryotes?To address this issue, we use an MCU-KO HEK293T line as our model system. Consistent with animal experiments, these cells have normal mitochondrial morphology, matrix [Ca2+], and ATP production via oxidative phosphorylation. Interestingly, we observed a slow Ca2+ uptake in MCU-KO mitochondria, operating at 5-10% of the MCU capacity, resistant to a potent MCU inhibitor Ru360. Furthermore, these mitochondria lose Na+-dependent Ca2+ release, due to decreased expression of the NCLX protein. We therefore propose that mitochondria compensate the loss of MCU by invoking a backup Ca2+ uptake mechanism, and by suppressing Ca2+ release. Supporting this hypothesis, we show that expressing NCLX in MCU-free mitochondria drastically reduces matrix [Ca2+], and abolishes >80% ATP output. Moreover, overexpression of WT-NCLX, but not a loss-of-function mutant S468A-NCLX, induces rapid death of MCU-KO cells.We’re now investigating how mitochondria invoke these compensatory mechanisms. Na+/Ca2+ exchange can be restored by introducing human MCU gene into MCU-KO cells, but not by expressing transport-capable MCU homologues from A. thaliana or D. Discoideum, implicating that mitochondria might balance Ca2+ uptake and release through an MCU-NCLX interaction that stabilizes inner-membrane expression of NCLX.