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

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 μM) 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 μM) had no significant effect on the decay of mitochondrial [Ca²⁺]. Inhibition of the mitochondrial Na⁺-Ca²⁺ exchanger with CGP-37157 (50 μM) 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.