Mechanistic basis of EMRE's essential role in the regulation of mitochondrial calcium uniporter complex

Mitochondrial Ca2+ uptake through the mitochondrial calcium uniporter complex (MCUcx) is a critical determinant of cellular metabolism, integrating Ca2+ signaling with ATP production and redox control. Yet how MCUcx activity is constrained to prevent Ca2+ overload and cell injury, and how the essential MCU regulator (EMRE), a subunit required for channel activity, mechanistically supports MCUcx function remains incompletely defined. Here, using a newly developed high-sensitivity assay to quantify MCUcx function in intact mitochondria, we uncover two fundamental roles of EMRE. First, EMRE is required for robust matrix Ca2+-dependent inhibition of MCUcx, acting through a juxtamembrane site via a mechanism distinct from MICU1-mediated inhibition at low cytosolic Ca2+. Second, by decoupling channel function from regulation, we demonstrate that EMRE promotes robust ion permeation through MCUcx, elevating its role from a structural scaffold to an active determinant of channel throughput. Together, our findings refine current models of mitochondrial Ca2+ regulation, establish EMRE as an essential multifunctional regulator of uniporter activity, and highlight the utility of our assay for probing MCUcx biophysical mechanisms and enabling the discovery of uniporter modulators. Significance StatementMitochondria use Ca2+ signals to adjust energy production to cellular demand, but excessive Ca2+ entry can trigger cell death. How the mitochondrial calcium uniporter balances these opposing needs remains fundamentally unresolved. Using a high-sensitivity approach that isolates uniporter permeation from Ca2+-dependent confounders in intact mitochondria, we characterize a matrix Ca2+-dependent inhibitory mechanism that depends on EMRE and is functionally distinct from MICU1-mediated regulation. We further show that EMRE, a small regulatory subunit unique to higher organisms, not only enables channel function but promotes robust ion permeation through the pore. Together, these findings refine current models of mitochondrial Ca2+ regulation and provide a unified framework for understanding EMRE-dependent uniporter regulation in intact mitochondria.