Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics.

Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration (Ca(2+)) is elevated to levels approaching approximately 500 nM, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked Ca(2+) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration (Ca) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low Ca, but high K(+) depolarization (50 mM, 45 sec), which elevates Ca(2+) to approximately 600 nM, increased Ca to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of Ca was a function of both depolarization strength and duration. After repolarization, Ca recovered to prestimulation levels with a time course that paralleled the decline in Ca(2+). Depolarization-induced increases in Ca were spatially heterogeneous. At the level of single mitochondria, Ca elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial Ca(2+) gradients that exist early during depolarization. Within individual mitochondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings demonstrate that in situ Ca accumulation by mitochondria, now directly identified as the structural correlate of the "FCCP-sensitive store, " is robust, reversible, graded with stimulus strength and duration, and dependent on spatial location.