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 similar to 320 muM). 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 similar to1 muM. 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. (C) 2003 Elsevier Science Ltd. All rights reserved.