Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

Mitochondrial Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter (MCU) following store-operated Ca²⁺ entry supports cellular bioenergetics, yet how mitochondria shape store-operated Ca²⁺ entry and cytosolic Ca²⁺ signaling remains incompletely understood. Combining gene deletion and functional Ca²⁺ imaging techniques with a rigorous transcriptomic filter, we find larger cytosolic Ca²⁺ signals in CRISPR/Cas9-generated Mcu KO cells. This increase arises primarily from slower cytosolic Ca²⁺ clearance rather than increased store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity. Compensatory upregulation of cytosolic Ca²⁺ regulators, such as the plasma membrane Ca²⁺ ATPase pump that extrudes excess cytosolic Ca²⁺, is insufficient to restore normal Ca²⁺ homeostasis. Reexpression of WT MCU restored the cytosolic Ca²⁺ dynamics but a channel pore-dead MCU mutant did not. Deletion of Mcu resulted in major alterations in the transcriptome and reexpression of the protein significantly restored 15% of more than 200 common genes that showed differential expression in two independent KO clones. Our results identify a set of candidate MCU-dependent genes that may contribute to the regulation of cellular Ca²⁺ signaling, and show how cytosolic Ca²⁺ signals can be enhanced in the absence of MCU without an increase in Ca²⁺ release-activated Ca²⁺ channel activity.