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

Several lines of evidence suggest that neuronal mitochondria accumulate cal cium when the cytosolic free Ca2+ concentration ([Ca2+](i)) is elevated to levels approaching similar to 500 nM, but the spatial, temporal, and quanti tative characteristics of net mitochondrial Ca uptake during stimulus-evoke d [Ca2+](i) elevations are not well understood. Here, we report direct meas urements of depolarization-induced changes in intramitochondrial total Ca c oncentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibite d undetectably low [Ca](mito), but high K+ depolarization (50 mM, 45 sec), which elevates [Ca2+](i) to similar to 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanid e p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolari zation, [Ca](mito) recovered to prestimulation levels with a time course th at paralleled the decline in [Ca2+](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondr ia, [Ca](mito) elevations depended on proximity to the plasma membrane, con sistent with predictions of a diffusion model that considers radial [Ca2+]( i) gradients that exist early during depolarization. Within individual mito chondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings de monstrate that in situ Ca accumulation by mitochondria, now directly identi fied as the structural correlate of the "FCCP-sensitive store," is robust, reversible, graded with stimulus strength and duration, and dependent on sp atial location.