Mitochondria have a very large capacity to accumulate Ca2+ during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca2+]M (mitochondrial [Ca2+]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca2+]M during prolonged stimulation has been previously precluded by the high Ca2+ affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca2+] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca2+ to permeabilized HeLa cells triggers an increase in [Ca2+]M up to an steady state of approximately 2–3 mM in the absence of phosphate and 0.5–1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca2+] reaches 0.5–1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca2+]M levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca2+ uniporter largely reduced the rate of [Ca2+]M increase, but the final steady-state [Ca2+]M reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca2+]M without problems derived from aequorin consumption.

You do not currently have access to this content.