Quantitative estimate of mitochondrial [Ca2+] in stimulated motor nerve terminals.
Peak values reported for mitochondrial matrix [Ca(2+)] 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 [Ca(2+)] approaches millimolar levels. To avoid this potential problem, we loaded lizard motor terminal mitochondria with the low-affinity indicator rhod-5N (K(d) approximately 320 microM). During trains of action potentials at 50Hz, matrix fluorescence transients (measured as F/F(rest)) increased to a plateau level that was maintained throughout the stimulus train. This plateau of matrix [Ca(2+)] occurred in spite of evidence that Ca(2+) continued to enter the terminal and continued to be sequestered by mitochondria. When the stimulation frequency was increased, or when Ca(2+) entry per action potential was increased with the K(+) channel blocker 3,4-diaminopyridine (3,4-DAP), or reduced by lowering bath [Ca(2+)], the rate of rise of matrix [Ca(2+)] changed, but the plateau amplitude remained constant. Calculations demonstrated that the F/F(rest) measured at this plateau corresponded to a matrix [Ca(2+)] of approximately 1 microM. The high K(d) of rhod-5N ensures that this value is not a result of dye saturation, but rather reflects a powerful Ca(2+) buffering mechanism within the matrix of these mitochondria.
David
Gavriel
G
Department of Physiology and Biophysics, University of Miami School of Medicine, R-430, P.O. Box 016430, Miami, FL 33101, USA. gdavid@newssun.med.miami.edu
Talbot
Janet
J
Barrett
Ellen F
EF
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000172:deficiency*; D020013:Calcium Signaling / Q000502:physiology*; D004558:Electric Stimulation; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008116:Lizards; D008928:Mitochondria / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000294:innervation / Q000502:physiology; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000648:ultrastructure; D009924:Organ Culture Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D012235:Rhodamines; D009435:Synaptic Transmission / Q000502:physiology
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D026902:Potassium Channel Blockers; C574804:Rhod-5N; D012235:Rhodamines; D002118:Calcium