Membrane current evoked by mitochondrial Na+–Ca2+ exchange in mouse heart

Figures

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  Fig. 1. Quality assessment of mitochondria. a Western blot analyses of mitochondria isolated from mouse ventricle. Mitochondrial identity was confirmed by assessing the expressions of MCU and COX IV. b A mitoplast stained with MitoTracker Green. Transmit, fluorescent and merged images of a mitoplast are shown. A bar indicates 2 μm

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  Fig. 2. Na+-induced Ca2+ efflux from isolated mitochondria. a Representative traces of mitochondrial Ca2+ uptake assay. Blocking Ca2+ influx through MCU by 5 μM Ru360 induced Ca2+ efflux from mitochondria, which was accelerated by extra-mitochondrial Na+ (25 mM). b Initial Ca2+ efflux rate in the absence and presence of extra-mitochondrial Na+ (n = 4). *p < 0.05

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  Fig. 3. Na+-induced inward current. a Membrane currents in the absence (left) and presence (middle) of 50 mM Na+ in the bath solution. The difference currents, middle minus left, are shown in the right. b Current–voltage relationships in the absence (black) and presence (red) of 50 mM Na+ in the bath solution (n = 4). The amplitude was measured near the end of square pulses. c The difference currents derived from b (n = 4). Square pulse protocol was used in a–c. d Representative current–voltage relationships in the absence (black) and presence (red) of 50 mM Na+ in the bath solution. e The difference current derived from d. Ramp voltage pulse protocol was used in d and e. The pipette solution contained 1 μM free Ca2+ in all experiments

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  Fig. 4. Inhibition of Na+- or Li+-induced inward current by CGP-37157. a Current–voltage relationships of 50 mM Na+-induced current in the absence (black) and presence (red) of 2 μM CGP-37157 (n = 4). b The current amplitude at −150 mV. c Current–voltage relationships of 50 mM Li+-induced current in the absence (black) and presence (red) of 2 μM CGP-37157 (n = 4). d The current amplitude at −150 mV. In all experiments, pipette solution contained 1 μM free Ca2+. *p < 0.05. CGP: CGP-37157

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  Fig. 5. Na+- and Ca2+-concentration dependences of Na+-induced inward current. a Current–voltage relationships of Na+-induced current with extra-mitochondrial 12.5, 25 and 50 mM Na+ (n = 5). b Extra-mitochondrial Na+ concentration dependence of Na+-induced inward current. Current was measured at −150 mV and normalized to the current induced by 50 mM Na+. Fitted half-maximum concentration and Hill coefficient are 35.6 mM and 3.7, respectively. c Current–voltage relationships of 50 mM Na+-induced current with 0, 1 and 10 μM Ca2+ in the pipette solution (n = 6–15). d Intra-mitochondrial Ca2+ concentration dependence of inward current at −150 mV. **p < 0.01 vs current without Ca2+ in pipette

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  Fig. 6. Extra-mitochondrial Ca2+-induced current. Current–voltage relationships in the absence and presence of 1 mM Ca2+ in the bath solution (n = 4). 1 mM extra-mitochondrial Ca2+ with 100 mM Na+ in the matrix side did not induce outward current

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  Fig. 7. Ca2+ uptake through reverse mode of NCXm in isolated mitochondria. a, b Ca2+ uptake in the depolarized mitochondria. Intra-mitochondrial Ca2+ was measured in Na+-unloaded mitochondria (0 Na+), Na+-loaded mitochondria (Na+-loaded) and Na+-loaded mitochondria with 20 μM CGP-37157 (Na+-loaded + CGP). a Representative data. b Initial rates of intra-mitochondrial Ca2+ rise (n = 4). *p < 0.05. c, d Ca2+ uptake in energized mitochondria. Intra-mitochondrial Ca2+ uptake was measured in control (control), in the presence of Ru360 (+ Ru360) and in the presence of ruthenium red (+ RuR). c Representative data. d Initial rates of intra-mitochondrial Ca2+ rise (n = 4). *p < 0.05