Mitochondrial free [Ca2+] levels and the permeability transition

Mitochondrial Ca²⁺ activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca²⁺] ([Ca²⁺]_M) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca²⁺]_M from increasing above 2–3 μM. Instead, using low-Ca²⁺-affinity aequorin probes, we have measured [Ca²⁺]_M values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca²⁺]_M to levels of 0.5–1 mM was actually observed at any phosphate concentration (0–10 mM) during continuous perfusion of 3.5–100 μM Ca²⁺-buffers. In spite of this high and maintained (>10 min) [Ca²⁺]_M, mitochondria retained functionality and the [Ca²⁺]_M drop induced by a protonophore was fully reversible. In addition, this high [Ca²⁺]_M did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca²⁺]_M. In conclusion [Ca²⁺]_M levels of 0.5–1 mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.