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Quantitative estimate of mitochondrial [Ca2+] in stimulated motor nerve terminals
Gavriel David1, Janet Talbot, Ellen F Barrett
1Department 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
Abstract:
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.
Insights
Mitochondrial matrix calcium levels in lizard motor terminals were measured using a low-affinity dye, revealing a stable plateau of approximately 1 microM during stimulation. This indicates a potent mitochondrial calcium buffering system, unaffected by changes in calcium influx or stimulation frequency.
Area of Science:
- Cellular Biology
- Neuroscience
- Mitochondrial Physiology
Background:
- Mitochondrial matrix calcium ([Ca(2+)]) measurements vary widely, from micromolar to millimolar.
- High-affinity fluorescent dyes risk saturation at high calcium concentrations, potentially skewing results.
Purpose of the Study:
- To accurately measure mitochondrial matrix calcium during stimulation in lizard motor terminals.
- To investigate the buffering capacity of mitochondria under varying calcium loads.
Main Methods:
- Utilized the low-affinity fluorescent indicator rhod-5N (K(d) ≈ 320 µM) to avoid dye saturation.
- Stimulated lizard motor terminals with action potentials at 50Hz.
- Manipulated extracellular calcium and used 3,4-diaminopyridine to alter calcium influx.
Main Results:
- Matrix calcium fluorescence reached a stable plateau during 50Hz stimulation, irrespective of altered calcium entry.
- The plateau level corresponded to approximately 1 µM matrix calcium, confirmed by calculations.
- This stable plateau persisted even when stimulation frequency or calcium influx was modified.
Conclusions:
- Lizard motor terminal mitochondria possess a powerful endogenous calcium buffering mechanism.
- The buffering system maintains matrix calcium at a stable ~1 µM level, preventing saturation of low-affinity indicators.
- This buffering is a critical factor in regulating mitochondrial function during neuronal activity.