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, 104 (11), 2353-61

Mitochondrial Free Ca²⁺ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

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Mitochondrial Free Ca²⁺ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Maxim V Ivannikov et al. Biophys J.

Abstract

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (ΔΨm), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pHm) and ΔΨm were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]m was 0.22 ± 0.04 μM, but it rose to ~26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]m coincided with a rapid elevation in pHm and was followed by an after-stimulus ΔΨm hyperpolarization. However, pHm decreased and no ΔΨm hyperpolarization was observed in response to lower levels of [Ca²⁺]m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]m are required to stimulate presynaptic mitochondrial energy metabolism.

Figures

Figure 1
Figure 1
Imaging of mitochondrial pH and Ca2+ changes in Drosophila larval motor neuron terminals. (A) Images of a motor neuron forming a terminal with type-Ib big boutons on the surface of muscle fiber 13. Top to bottom: phase-contrast image; boutons containing cytosolic (cyto.) GFP; mitochondria (mito.) loaded with rhod-FF; merge of cyto. and mito. (B) Changes in cytosolic Ca2+, [Ca2+]i (blue line; F/Fo, GCaMP3), mitochondrial matrix Ca2+, [Ca2+]m (black traces; (2Fo − F)/Fo, mito-RP excitation at 420 nm), and pHm (red traces, F/Fo, mito-RP excitation at 490 nm) obtained upon 80 Hz nerve stimulation for 2 s at 10 s in normal (upper), Ca2+-free (middle), and 50 μM bongkrek acid (BA) containing HL6 solution (lower).
Figure 2
Figure 2
Quantifying mitochondrial matrix Ca2+ concentrations with rhod-FF and rhod-5N indicators. (A) Fluorescent responses of rhod-FF (black) and rhod-5N (red) normalized to 80 Hz recorded from presynaptic mitochondria in MN13-Ib axonal terminals stimulated with 42- and 80-Hz electrical pulse trains for 2 s at 10 and 20 s. Dotted lines show the 42/80-Hz ratio values for both indicators. (B) Graphical simulation of rhod-FF and rhod-5N fluorescence (F/Fmax, Fmin = 0) at different Ca2+ concentrations ([Ca2+]) for a range of Kd values for both indicators (thick, 1 × Kd, to thin, 1.5 × Kd). Dotted lines show the rhod-FF and rhod-5N fluorescence intensities that give 42/80-Hz ratios similar to those in A and also result in single numerical solutions for [Ca2+] for rhod-FF and rhod-5N at 42 and 80 Hz. Dotted areas are the solution spaces for [Ca2+] at 42 and 80 Hz for various Kd.
Figure 3
Figure 3
Measuring mitochondrial matrix Ca2+ concentrations with the genetically encoded Ca2+ indicator mito-D4cpv. (A) Calibration curve (four-parameter Hill’s fit, R2 = 0.95) showing EYEP(cpVenus)/ECFP ratio of mito-D4cpv as a function of the logarithm of Ca2+ concentration (μM). (B) Changes in EYFP (red line) and ECFP fluorescence (black line) and their ratio (dotted blue line) in mitochondria of M13-1b axonal terminals stimulated with 42- and 80-Hz electrical pulse trains for 2 s at 10 and 20 s.
Figure 4
Figure 4
Interrelation between mitochondrial matrix Ca2+, cytosolic Ca2+, and mitochondrial matrix pH. (A) Graph showing a linear relationship between the volume average cytosolic Ca2+ concentration, [Ca2+]i, and mitochondria matrix Ca2+ concentration, [Ca2+]m. (B) (Left) Graph depicting the equilibrium mitochondrial matrix pH associated with different mitochondrial matrix Ca2+ concentrations, [Ca2+]m. (Right) Plot showing differential changes in mitochondrial matrix pH associated with ∼10 μM step increase in mitochondrial matrix Ca2+ concentration. Error bars indicate the mean ± SE.
Figure 5
Figure 5
Imaging of mitochondrial membrane potential changes (ΔΨm) with TMRE probe. (Upper) Representative traces of TMRE fluorescent changes ((F − Fo)/Fo = ΔF/Fo) in response to 2-s stimuli at 30 and 42 Hz in mitochondria of MN13-Ib terminals. (Lower) Quantification of after-stimulus ΔΨm increases for different stimulation frequencies. Multigroup comparisons were carried out using analysis of variance with Tukey’s posttest. Error bars indicate the mean ± SE.

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