Peak values reported for mitochondrial matrix [Ca2+] following stimulation have ranged from micromolar to near-millimolar in various cells. Measurements using fluorescent indicators have traditionally used high-affinity dyes such as rhod-2, whose fluorescence would be expected to saturate if matrix [Ca2+] approaches millimolar levels. To avoid this potential problem, we loaded lizard motor terminal mitochondria with the low-affinity indicator rhod-5N (Kd ∼ 320 μM). During trains of action potentials at 50 Hz, matrix fluorescence transients (measured as F/Frest) increased to a plateau level that was maintained throughout the stimulus train. This plateau of matrix [Ca2+] occurred in spite of evidence that Ca2+ continued to enter the terminal and continued to be sequestered by mitochondria. When the stimulation frequency was increased, or when Ca2+ entry per action potential was increased with the K+ channel blocker 3,4-diaminopyridine (3,4-DAP), or reduced by lowering bath [Ca2+], the rate of rise of matrix [Ca2+] changed, but the plateau amplitude remained constant. Calculations demonstrated that the F/Frest measured at this plateau corresponded to a matrix [Ca2+] of ∼ 1 μM. The high Kd of rhod-5N ensures that this value is not a result of dye saturation, but rather reflects a powerful Ca2+ buffering mechanism within the matrix of these mitochondria.
- Fluorescent indicators
- Mitochrondrial [Ca]
- Stimulated motor nerve terminals
ASJC Scopus subject areas
- Cell Biology