Depolarization-Induced Mitochondrial Ca Accumulation in Sympathetic Neurons: Spatial and Temporal Characteristics

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Abstract
Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca2+concentration ([Ca2+]i) is elevated to levels approaching ∼500 nm, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked [Ca2+]ielevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrialtotal 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 [Ca2+]i to ∼600 nm, increased [Ca]mito to 13.0 ± 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanidep-(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 [Ca2+]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 [Ca2+]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.