The Mitochondrial Ca²⁺ uniporter is a central regulator of interorganellar Ca²⁺ transfer and NFAT activation

Mitochondrial Ca²⁺ uptake tailors the strength of stimulation of plasma membrane phospholipase C–coupled receptors to that of cellular bioenergetics. However, how Ca²⁺ uptake by the mitochondrial Ca²⁺ uniporter (MCU) shapes receptor-evoked interorganellar Ca²⁺ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca²⁺ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca²⁺ signaling across mammalian cell types. MCU activity sustains cytosolic Ca²⁺ signaling by preventing Ca²⁺-dependent inactivation of store-operated Ca²⁺ release–activated Ca²⁺ channels and by inhibiting Ca²⁺ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca²⁺ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca²⁺ oscillations, endoplasmic reticulum Ca²⁺ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol–mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca²⁺ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca²⁺ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca²⁺ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca²⁺ signals that regulate cellular transcription and function.