Extrusion of Ca2+ from mouse motor terminal mitochondria via a Na+-Ca2+ exchanger increases post-tetanic evoked release

Abstract

Mitochondria sequester much of the Ca2+ that enters motor nerve terminals during repetitive stimulation at frequencies exceeding 10-20 Hz. We studied the post-stimulation extrusion of Ca2+ from mitochondria by measuring changes in matrix [Ca2+] with fluorescent indicators loaded into motor terminal mitochondria in the mouse levator auris longus muscle. Trains of action potentials at 50 Hz produced a rapid increase in mitochondrial [Ca2+] followed by a plateau, which was usually maintained after the end of the stimulus train and then slowly decayed back to baseline. Increasing the Ca2+ load delivered to the terminal by increasing the number of stimuli (from 500 to 2000) or the stimulation frequency (from 50 to 100 Hz), by increasing bath [Ca2+], or by prolonging the action potential with 3,4-diaminopyridine (100 microM) prolonged the post-stimulation decay of mitochondrial [Ca2+] without increasing the amplitude of the plateau during stimulation. Inhibiting the opening of the mitochondrial permeability transition pore with cyclosporin A (5 microM) had no significant effect on the decay of mitochondrial [Ca2+]. Inhibition of the mitochondrial Na+-Ca2+ exchanger with CGP-37157 (50 microM) dramatically prolonged the post-stimulation decay of mitochondrial [Ca2+], reduced post-stimulation residual cytosolic [Ca2+], and reduced the amplitude of endplate potentials evoked after the end of a stimulus train in the presence of both low and normal bath [Ca2+]. These findings suggest that Ca2+ extrusion from motor terminal mitochondria occurs primarily via the mitochondrial Na+-Ca2+ exchanger and helps to sustain post-tetanic transmitter release at mouse neuromuscular junctions.

Figure 1. Localization of stimulation-induced increases in X-rhod-5F fluorescence within a yellow fluorescent protein-expressing mouse motor nerve terminal

A, overlay of phase and yellow fluorescent protein (YFP; green) images. B, overlay of phase and X-rhod-5F difference (red) images, illustrating regions in which fluorescence increased in response to 50-Hz stimulation for 20 s. The difference image was created by subtracting the average prestimulation fluorescence (20 frames) from the average fluorescence during stimulation (six frames). C, overlay of YFP image and X-rhod-5F difference image. Calibration bar, 50 μm. D, time course of stimulation-induced X-rhod-5F fluorescence changes (plotted as F/F_rest = F_net/F_rest) in this terminal. The dashed horizontal line indicates the baseline measured from prestimulation images. Dashed vertical lines indicate the duration of stimulation. For this record, as well as those in Figs 2–6, muscle contraction was blocked using d-tubocurarine (10 μg/ml). The plateau amplitude observed during and immediately following the stimulus train in this and subsequent figures is not an artifact due to dye saturation. As noted in the Introduction, the maximal stimulation-induced increase in matrix [Ca²⁺] in normal motor terminals is only 1–2 μm above a resting level estimated as 0.05–0.10 μm, and none of the rhod dyes used here would saturate over this range. Also, a similar stimulation-induced plateau of matrix [Ca²⁺] is evident with a very low affinity indicator (rhod-5N, K_d∼300 μm, David et al. 2003).

Figure 2. Increasing the duration of a 50-Hz stimulus train prolongs the post-stimulation decay of mitochondrial [Ca²⁺] measured by changes in the fluorescence of X-rhod-5F

A, two trains (1000 and 2000 stimuli) were delivered sequentially to a single terminal. Curves drawn through the data points were calculated as the weighted average of the five nearest neighbours. The dotted vertical lines mark (from left to right) the beginning of the 2000 stimulus train, the beginning of the subsequent 1000 stimulus train, and the end of stimulation for both trains. B, pairwise comparison of the time integrals of the post-train decays of mitochondrial [Ca²⁺] measured after 1000 stimuli (n = 4 terminals) or 2000 stimuli (n = 3 terminals), normalized to those measured in the same terminal after 500 stimuli.

Figure 3. The post-stimulation decay of mitochondrial [Ca²⁺] is accelerated by reducing bath [Ca²⁺] (A) and slowed by 3,4-diaminopyridine (3,4-DAP) (B)

A, responses to trains of 500 stimuli at 50 Hz measured in a motor terminal in a bath [Ca²⁺] of 1.8 and 0.4 mm. Similar changes were seen in another terminal (not shown) in which bath [Ca²⁺] was changed from 2.4 to 0.4 mm. B, responses to trains of 1000 stimuli at 50 Hz measured in another terminal before and 24 min after adding 100 μm 3,4-DAP. The constant amplitude of the peak increase in mitochondrial [Ca²⁺] in different bath [Ca²⁺] and after addition of 3,4-DAP is consistent with the hypothesized formation of an insoluble calcium-containing complex that limits the increase in matrix [Ca²⁺] (see Introduction). X-rhod-1 was used in both experiments.

Figure 4. Increasing the frequency of stimulation prolongs the post-stimulation decay of mitochondrial [Ca²⁺] measured using rhod-2

Six trains of 1000 stimuli were delivered sequentially to a single terminal, three at 50 Hz and three at 100 Hz, alternating the frequencies. The averages of each set of three trains are shown.

Figure 5. The mitochondrial Na⁺–Ca²⁺ exchanger contributes more to mitochondrial Ca²⁺ extrusion than openings of the mitochondrial permeability transition pore

A, responses to 500 stimuli at 50 Hz recorded in a single terminal before and 22 min after addition of 50 μm CGP-37157, which blocks the mitochondrial Na⁺–Ca²⁺ exchanger. B, responses to 1000 stimuli at 50 Hz recorded in a different terminal before and 18 min after addition of 5 μm cyclosporin A, which inhibits some openings of the permeability transition pore. X-rhod-1 was used in both experiments.

Figure 6. Residual cytosolic [Ca²⁺] after a tetanus is reduced after inhibition of the mitochondrial Na⁺–Ca²⁺ exchanger with CGP-37157

Cytosolic [Ca²⁺] was monitored as F/F_rest for OG-5N (A) or OG-1 (B and C) loaded ion ophoretically into the axon. Each record shows the average of three to four stimulus trains recorded in the same terminal before and 15–80 min after addition of 50 μm CGP-37157; 500 stimuli in A and B; 1000 stimuli in C. A–C were from different terminals, all in 1.8 mm bath Ca²⁺. Error bars indicate s.e.m.

Figure 7. Inhibition of mitochondrial Ca²⁺ extrusion with CGP-37157 reduces post-tetanic evoked release in near-physiological conditions (A), with reduced temperature and fewer stimuli (B) and in low bath [Ca²⁺] (C)

A–C, EPP amplitudes (normalized to EPP amplitude before the train) during and after 50-Hz stimulation. Error bars indicate s.d. Insets in the lower right of A and C show the difference between post-tetanic EPP amplitudes recorded in the absence and presence of CGP-37157. Mono-exponential decays (solid line) fitted to these data had time constants of 10.5 s in A (95% confidence interval, 8.8–12.1 s) and 22.6 s in C (95% confidence interval, 9.8–35 s). D, shows tetanic EPPs from C on an expanded time scale to show effects of CGP-37157 during the train. Curves are the weighted average of nine nearest neighbours. In all experiments muscle contractions were blocked with 2.5–4 μmμ-conotoxin GIIIB. Data in A were averaged from 17 trains recorded from 12 endplates in control (○), and from 12 trains in 10 terminals after addition of CGP-37157 (•). Data in B are the average of three control and six post-drug trains from the same terminal. Data in C and D are the average of 12 trains from 12 terminals in four animals (control) and 10 trains from 10 terminals (drug).