Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 197-206 |
| Number of pages | 10 |
| Journal | Cell Calcium |
| Volume | 33 |
| Issue number | 3 |
| DOIs | |
| State | Published - Mar 1 2003 |
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Keywords
- Fluorescent indicators
- Mitochrondrial [Ca]
- Stimulated motor nerve terminals
ASJC Scopus subject areas
- Cell Biology
- Endocrinology
Cite this
Quantitative estimate of mitochondrial [Ca2+] in stimulated motor nerve terminals. / David, Gavriel; Talbot, Janet; Barrett, Ellen.
In: Cell Calcium, Vol. 33, No. 3, 01.03.2003, p. 197-206.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Quantitative estimate of mitochondrial [Ca2+] in stimulated motor nerve terminals
AU - David, Gavriel
AU - Talbot, Janet
AU - Barrett, Ellen
PY - 2003/3/1
Y1 - 2003/3/1
N2 - 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.
AB - 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.
KW - Fluorescent indicators
KW - Mitochrondrial [Ca]
KW - Stimulated motor nerve terminals
UR - http://www.scopus.com/inward/record.url?scp=0037339320&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037339320&partnerID=8YFLogxK
U2 - 10.1016/S0143-4160(02)00229-4
DO - 10.1016/S0143-4160(02)00229-4
M3 - Article
VL - 33
SP - 197
EP - 206
JO - Cell Calcium
JF - Cell Calcium
SN - 0143-4160
IS - 3
ER -