Inorganic polyphosphate (polyP) is required for sustained free mitochondrial calcium elevation, following stimulated calcium uptake.

Mitochondrial free calciumis critically linked to the regulation of bioenergeticsand cellular signaling. Free calciumconcentrations within the organelleare regulated by two processes: flux across the mitochondrial inner membranesand buffering by phosphate. The key role of phosphate in the buffering of free calciumin mitochondria is well-established. Specifically, during stimulated calciumuptake, calciumis partially buffered by orthophosphate, allowing for elevated calciumconcentrations, while preventing calciumtoxicity. However, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calciumrequires the presence of free inorganic polyphosphate (polyP) within the organelle. Specifically, we found that the verexpression of a mitochondrial targeted enzyme hydrolyzing polyP, leads to the loss of the cellular ability to maintain elevated calciumconcentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calciumphosphate insoluble clusters, allowing for the maintenance of elevated free calciumlevels, during stimulated calciumuptake.