biostudies-literatureIvannikov MVNIA NIH HHSNINDS NIH HHS2353-61https://www.ebi.ac.uk/biostudies/studies/S-EPMC3672877biostudies-literatureUnknown104(11)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.Biophysical journalMitochondrial free Ca²⁺ levels and their effects on energy metabolism in Drosophila motor nerve terminals.PMC3672877T32 AG021890R01 NS061914Ivannikov MVMacleod GTfalseMitochondrial free Ca²⁺ levels and their effects on energy metabolism in Drosophila motor nerve terminals.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.2013-01-01T00:00:00Z2013 Jun2025-04-21T14:35:09.636Z2019-03-27T01:10:50ZS-EPMC36728772374650710.1016/j.bpj.2013.03.064