Kumari, A.; Nguyen, D. M.; Disilvestre, D.; Dirda, N. D. A.; Kethanapalli, S. H.; Kao, J. P. Y.; Garg, V.

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.