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

• Published in: Biophysical journal Vol. 104; no. 11; pp. 2353 - 2361
• Main Authors: Ivannikov, Maxim V., Macleod, Gregory T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.06.2013; The Biophysical Society
• Subjects: Animals; Axons; calcium; Calcium - metabolism; Cell Biophysics; Drosophila; Drosophila melanogaster - cytology; Energy Metabolism; enzymes; Female; larvae; Male; membrane potential; mitochondria; Mitochondria - metabolism; Motor Neurons - cytology; nerve tissue; neurons; oxidative phosphorylation
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2013.03.064

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.