title: Mitochondrial free Ca²⁺ levels and their effects on energy metabolism in Drosophila motor nerve terminals.
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.
authors.affiliation: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. ivannikov@uthscsa.edu
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Ivannikov
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Macleod
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology*; D004734:Energy Metabolism; D005260:Female; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D009046:Motor Neurons / Q000166:cytology*
chemical_list: D002118:Calcium