Matrix polyamines regulate bidirectional calcium flux through MCU

Polyamines, well-known regulators of the mitochondrial calcium (Ca²⁺) uniporter channel, show unexpected effects when binding the channel from within the matrix. Using cryo-EM, molecular dynamics simulations, and mutagenesis experiments, we determine that polyamines achieve such regulation by binding within the pore to a ring of negative residues forming a matrix gate, inhibiting Ca²⁺ conduction. In whole-mitoplast electrophysiology assays, matrix polyamines cause a gradual increase in Ca²⁺ currents during prolonged conduction, due to relief of this inhibition. Notably, this electrostatic binding increases 3-fold as the inner membrane depolarizes, preventing Ca²⁺ efflux. Additionally, we also identify that phospholipids form part of the Ca²⁺ conduction pathway through MCU. Because we find significant variability in matrix polyamine content across mouse organs, this unexpected mechanism for sculpting the mitochondrial Ca²⁺ waveform suggests a tissue-specific regulation of metabolism.