Mitochondrial clearance of cytosolic Ca²⁺ in stimulated lizard motor nerve terminals proceeds without progressive elevation of mitochondrial matrix [Ca²⁺]

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.