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

Gavriel David, Janet Talbot, Ellen F. Barrett

Research output: Contribution to journalArticle

42 Scopus citations

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 languageEnglish (US)
Pages (from-to)197-206
Number of pages10
JournalCell Calcium
Volume33
Issue number3
DOIs
StatePublished - Mar 1 2003

Keywords

  • Fluorescent indicators
  • Mitochrondrial [Ca]
  • Stimulated motor nerve terminals

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

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