Mitochondrial free Ca²⁺ levels and their effects on energy metabolism in Drosophila motor nerve terminals.

Biophysical Journal Volume 104 June 2013 2353–2361 2353 Mitochondrial Free Ca2D Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals Maxim V. Ivannikov* and Gregory T. Macleod Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas ABSTRACT Mitochondrial Ca2þ uptake exerts dual effects on mitochondria. Ca2þ accumulation in the mitochondrial matrix dissipates membrane potential (DJm), but Ca2þ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca2þ ([Ca2þ]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca2þ]m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca2þ indicators with a 20-fold difference in Ca2þ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca2þ indicator (D4cpv) calibrated against known Ca2þ levels. Matrix pH (pHm) and DJm were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca2þ]m was 0.22 5 0.04 mM, but it rose to ~26 mM (24.3 5 3.4 mM with rhod-FF/rhod5N and 27.0 5 2.6 mM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca2þ]m coincided with a rapid elevation in pHm and was followed by an after-stimulus DJm hyperpolarization. However, pHm decreased and no DJm hyperpolarization was observed in response to lower levels of [Ca2þ]m, up to 13.1 mM. These data indicate that surprisingly high levels of [Ca2þ]m are required to stimulate presynaptic mitochondrial energy metabolism. INTRODUCTION Mitochondria provide most of the ATP that fuels a variety of cellular activities. Changes in cellular activities result in an increase in ATP demand and are usually associated with elevations in cytosolic ADP and calcium concentrations ([Ca2þ]i) (1,2), both of which are believed to modulate mitochondrial ATP synthesis. These regulatory mechanisms appear to be preserved in presynaptic nerve terminals, where the synchronization of ATP utilization with mitochondrial ATP production is crucial for sustaining synaptic transmission (3,4). An ADP stimulatory influence on energy metabolism has been well documented in isolated mitochondria and a number of neuronal preparations (1,2,5). The widely used ADP/ATP ratio reflects the energetic status of the cell and determines the amount of ADP available for phosphorylation by the F1-F0-ATP synthase (5). However, simulations of mitochondrial metabolism show that changes in the cytosolic ADP/ATP ratio and phosphate levels alone are not sufficient to explain mitochondrial metabolism stimulation (6). The calcium sensitivity of elements of mitochondrial energy metabolism provides an additional mechanism for stimulating ATP synthesis. An elevation in mitochondrial matrix Ca2þ levels ([Ca2þ]m) increases mitochondrial metabolism in motor nerve terminals in situ (7), although the [Ca2þ]m levels responsible for this stimulatory effect have not been quantified. Submitted November 27, 2012, and accepted for publication March 25, 2013. *Correspondence: Editor: David Piston. Ó 2013 by the Biophysical Society 0006-3495/13/06/2353/9 $2.00 Quantification of [Ca2þ]m levels that stimulate presynaptic mitochondrial energy metabolism in situ necessitates estimation of [Ca2þ]m along with several independent measures of mitochondrial energy metabolism, such as matrix pH (pHm) and mitochondrial membrane potential (DJm). The accuracy of any physiologically relevant [Ca2þ]m estimate measured with Ca2þ indicators depends on the indicator’s affinity, specificity of targeting to the matrix, environmental sensitivity, and accuracy of the calibration in situ, as well as the ability to replicate in vivo [Ca2þ]i transients. Either chemical or genetically encoded Ca2þ indicators (GECIs) can be used to measure [Ca2þ]m. GECIs offer subcellular specificity of targeting, but they are vulnerable to pH changes, and with the exception of aequorin, they have slow kinetics, nonlinear responses to Ca2þ, and a low dynamic range (8). Most chemical Ca2þ-indicators perform well in the areas in which GECIs are deficient, but they can be difficult to load with specificity, and few ratiometric chemical Ca2þ indicators are available with a Ca2þ affinity suitably low for measuring [Ca2þ]m. Nonratiometric imaging with dyes is particularly problematic, as their calibration generally requires permeabilization of the inner mitochondrial membrane to control [Ca2þ]m, which inevitably leads to dye loss (9). Exploiting the advantages of chemical Ca2þ indicators and GECIs in the same preparation limits negative influences imposed by their respective disadvantages, providing a greater degree of confidence in [Ca2þ]m estimates. The Drosophila larval neuromuscular preparation is amenable to the application of chemical Ca2þ indicators, and it is genetically tractable, allowing the expression of GECIs. http://dx.doi.org/10.1016/j.bpj.2013.03.064 2354 Further, the endogenous firing rates and accompanying changes in [Ca2þ]i have been quantified (7,10). Here, we use two different chemical Ca2þ indicators (rhod-5N, Kd ~ 320 mM, and rhod-FF, Kd ~ 19 mM) and adopt an analytical approach to calculate [Ca2þ]m from the ratio of their responses under similar conditions with no requirement for permeabilization. Two ratiometric GECIs, TN-XXL and D4cpv (Kd ~ 0.8 mM and Kd ~ 60 mM, respectively, in vitro) were used to estimate minimum and maximum [Ca2þ]m, respectively. A genetically encoded pH indicator (GEpHI; ratiometric pericam) and a mitochondrial potentiometric probe, tetramethylrhodamine ethyl ester (TMRE), were used to report elevation of mitochondrial energy metabolism. Both chemical and GECIs revealed that a high level of [Ca2þ]m (~26 mM) was required to stimulate presynaptic mitochondrial energy metabolism. Ivannikov and Macleod HL6 supplemented with 50 nM TMRE and the nerves were drawn into a stimulation pipette. TMRE-loaded larval preparations were stimulated at various frequencies with the dye present in the bath to minimize TMRE leakage. Dye and GECI fluorescence changes were imaged in MN13-Ib axonal terminals in abdominal segment 4, as described previously in Chouhan et al. (7). Briefly, nerve terminals were visualized using wide-field microscopy on a BX51WI microscope (Olympus, Center Valley, PA) equipped with a 100 (1.0 NA) water-immersion objective. Nerve stimulation at various frequencies was delivered via Master-8 stimulator (AMPI, Jerusalem, Israel) and a Digitimer (Brooksville, FL) model DS2A Mk.II. Images were captured with an Andor (Belfast, United Kingdom) iXonþ DU-860D EMCCD camera using Andor iQ 1.8 acquisition software and saved as 14-bit (128  128 pixels) (...truncated)