Differences in mitochondrial Ca 2+ handling during challenges of CaCl 2 pulses in brain synaptic and non‐synaptic mitochondria: implications for differential Ca 2+ buffering

Mitochondria play a key role in cytosolic Ca 2+ regulation and buffering, with Ca 2+ uptake mainly via the inner membrane mitochondrial Ca 2+ uniporter (MCU), and efflux of mitochondrial Ca 2+ largely via the Na + /Ca 2+ exchanger (mNCE). Previous reports have shown brain synaptic and non‐synaptic mitochondria display marked differences in Ca 2+ uptake before permeabilization of the mitochondrial membranes, the so‐called permeable transition pore (mPTP). However, the kinetics of mitochondrial Ca 2+ handling and its implications for Ca 2+ buffering after boluses of CaCl 2 have not been reported. In this study, we aimed to define the kinetics of Ca 2+ handling in synaptic and non‐synaptic mitochondria isolated from rat brains via discontinuous percoll centrifugation followed by differential centrifugation. Respiratory control index (RCI), and subsequent changes in calcium retention capacity (CRC) and membrane potential (ΔΨ m ), were assessed in isolated mitochondria. CRC and ΔΨ m were evaluated with Fura 4F penta‐K salt and TMRM dyes, respectively, using fluorescence spectrophotometry (Photon Technology). Mitochondria were energized with glutamate and malate, and CRC and ΔΨ m were assessed during state 2 respiration. To investigate the potential role of the mNCE in Ca 2+ handling, the NCE blocker CGP37157 (CGP) was used. In addition, experiments were conducted to determine the expression levels of the MCU and mNCE in the two mitochondria pools using western blot. Mitochondrial RCI was higher in the synaptic group compared to the non‐synaptic group, which is consistent with past results. Ca 2+ uptake and retention without CGP were lower in synaptic mitochondria compared to non‐synaptic mitochondria. Addition of CGP markedly enhanced CRC in the synaptic mitochondria to levels found in non‐synaptic mitochondria without CGP. In contrast, the CGP induced changes in CRC were not observed in the non‐synaptic mitochondria. Membrane potential depolarization occurred in both synaptic and non‐synaptic mitochondria as matrix free Ca 2+ accumulated, reflecting the decrease in Ca 2+ sequestration or Ca 2+ efflux in the non‐synaptic and synaptic mitochondria, respectively. MCU expression was higher in the non‐synaptic mitochondria compared to the synaptic mitochondria, whereas mNCE expression was not different. The increased Ca 2+ uptake and retention in the presence of CGP in the synaptic mitochondria implies that mNCE is active in synaptic mitochondria, which leads to faster extrusion of added Ca 2+ before it is sequestered. The lack of change in Ca 2+ kinetics in non‐synaptic mitochondria suggests that NCE activity is lower. The ΔΨ m and CRC results likely indicate that synaptic mitochondria can buffer Ca 2+ like that in non‐synaptic mitochondria, but only do so when the mNCE is blocked. We propose that synaptic mitochondria may have increased mNCE activity to regulate its matrix free Ca 2+ , while non‐synaptic mitochondria depend mostly on buffering of the free Ca 2+ ; the implications for these differential responses may be related to the physiological role of these two populations of mitochondria. Support or Funding Information Funding: NIH T35 HL072483 and MCW‐AHW This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .