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