Abstract
Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) 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+)](i) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low [Ca](mito), but high K(+) depolarization (50 mM, 45 sec), which elevates [Ca(2+)](i) to approximately 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolarization, [Ca](mito) recovered to prestimulation levels with a time course that paralleled the decline in [Ca(2+)](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondria, [Ca](mito) elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial [Ca(2+)](i) 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.