Abstract

Ca²⁺ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca²⁺ signaling. The primary pathway for mitochondrial Ca²⁺ uptake is the mitochondrial calcium uniporter (MCU), a Ca²⁺-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca²⁺ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca²⁺ uptake is tightly regulated to maintain low matrix [Ca²⁺] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca²⁺ regulation of intermembrane space-localized MICU1/2 is controlled by Ca²⁺-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca²⁺ that permeates through the channel pore regulates Ca²⁺ affinities of coupled inhibitory and activating sensors in the matrix. Ca²⁺ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca²⁺ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca²⁺ regulation of channel activity. Our results demonstrate how Ca²⁺ influx into mitochondria is tuned by coupled Ca²⁺-regulatory mechanisms on both sides of the inner mitochondrial membrane.