Quantitative estimate of mitochondrial [Ca²⁺] in stimulated motor nerve terminals

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 (K_d ∼ 320 μM). During trains of action potentials at 50 Hz, matrix fluorescence transients (measured as F/F_rest) 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/F_rest measured at this plateau corresponded to a matrix [Ca²⁺] of ∼ 1 μM. The high K_d 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.