Mitochondrial Clearance of Cytosolic Ca2+in Stimulated Lizard Motor Nerve Terminals Proceeds without Progressive Elevation of Mitochondrial Matrix [Ca2+]

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Abstract
This study used fluorescent indicator dyes to measure changes in cytosolic and mitochondrial [Ca2+] produced by physiological stimulation of lizard motor nerve terminals. During repetitive action potential discharge at 10–50 Hz, the increase in average cytosolic [Ca2+] reached plateau at levels that increased with increasing stimulus frequency. This stabilization of cytosolic [Ca2+] was caused mainly by mitochondrial Ca2+ uptake, because drugs that depolarize mitochondria greatly increased the stimulation-induced elevation of cytosolic [Ca2+], whereas blockers of other Ca2+ clearance routes had little effect. Surprisingly, during this sustained Ca2+ uptake the free [Ca2+] in the mitochondrial matrix never exceeded a plateau level of ∼1 μm, regardless of stimulation frequency or pattern. When stimulation ceased, matrix [Ca2+] decreased over a slow (∼10 min) time course consisting of an initial plateau followed by a return to baseline. These measurements demonstrate that sustained mitochondrial Ca2+ uptake is not invariably accompanied by progressive elevation of matrix free [Ca2+]. Both the plateau of matrix free [Ca2+] during stimulation and its complex decay after stimulation could be accounted for by a model incorporating reversible formation of an insoluble Ca salt. This mechanism allows mitochondria to sequester large amounts of Ca2+ while maintaining matrix free [Ca2+] at levels sufficient to activate Ca2+-dependent mitochondrial dehydrogenases, but below levels that activate the permeability transition pore.