MICU1-dependent Threshold and Cooperativity of Mitochondrial Ca2+ Uptake in the Liver

Recent studies have revealed MCU as the pore forming domain and MICU1 as a critical Ca2+-sensitive regulator of the mitochondrial Ca2+ uniporter. However, the mechanism of the complex Ca2+ dependence of the uniporter activity remains elusive. Our previous studies showed that prolonged down-regulation of MICU1 in HeLa cells causes lower threshold and decreased cooperativity of mitochondrial Ca2+ uptake. To study the functional significance of the effects of MICU1 we used hepatocytes harvested from the liver of mice exposed to in vivo silencing (4 weeks). Silencing of MICU1 or MCU resulted in >80% decrease in their respective mRNA levels. Silencing of MICU1 caused a leftward-shifted dose response and decreased cooperativity of mitochondrial Ca2+ uptake in both permeabilized and intact hepatocytes. By contrast, silencing of MCU resulted in slower Ca2+ uptake in the entire range of Ca2+ concentrations without change in threshold. Mitochondrial respiration and cellular ATP content were unaffected in media containing both glycolytic and mitochondrial fuels in either MICU1 or MCU-deficient hepatocytes. However, silencing of MICU1 caused an augmented loss of ATP when the cells were confined to oxidative metabolism and an enhanced sensitivity to mitochondrial Ca2+ overload and permeabilization. During stimulation with vasopressin, a Ca2+ mobilizing hormone, both MICU1 and MCU-deficient cells displayed an attenuated mitochondrial matrix [Ca2+] increase and stimulation of respiration. Collectively, these results show that keeping the gate of MCU closed by MICU1 at low [Ca2+] is required to maintain healthy mitochondria, and MICU1-mediated control of MCU (cooperativity?) is required to support the propagation of short-lasting calcium spikes and oscillations to the mitochondria and the ensuing physiological stimulation of oxidative metabolism.