Mitochondrial free [Ca2+] levels and the permeability transition.

Area of Science:

• Mitochondrial physiology
• Cellular calcium signaling

Background:

• Mitochondrial calcium (Ca2+) plays a crucial role in cellular processes, including metabolism and apoptosis.
• There is ongoing debate regarding the actual free mitochondrial Ca2+ ([Ca2+](M)) levels achievable in cells and mitochondria.

Purpose of the Study:

• To resolve controversies surrounding mitochondrial Ca2+ levels.
• To investigate the impact of high mitochondrial Ca2+ on mitochondrial function and permeability transition pore (PTP) opening.

Main Methods:

• Utilized low-Ca2+-affinity aequorin probes for measuring mitochondrial Ca2+.
• Performed direct in situ calibration of mitochondrial aequorin.
• Exposed mitochondria to varying Ca2+ and phosphate concentrations.
• Assessed mitochondrial functionality and PTP opening in response to Ca2+ and activators like phenyl arsine oxide (PAO).

Main Results:

• Measured sustained mitochondrial Ca2+ ([Ca2+](M)) levels of 0.5-1mM across a range of phosphate concentrations (0-10mM).
• Demonstrated that mitochondria maintain functionality and Ca2+ regulation at these elevated levels.
• Showed that high [Ca2+](M) alone does not induce PTP opening; additional activators (PAO) are required.
• Observed rapid and irreversible Ca2+ drop upon PAO addition, indicating PTP opening.

Conclusions:

• Mitochondria can achieve and sustain high free Ca2+ levels (0.5-1mM) for extended periods (>10 min) in the presence of phosphate.
• Massive opening of the permeability transition pore (PTP) is dependent on additional activators beyond elevated mitochondrial Ca2+.