Mitochondria buffer non-toxic calcium loads and release calcium through the mitochondrial permeability transition pore and sodium/calcium exchanger in rat basal forebrain neurons

Abstract

Mitochondria participate in intracellular Ca 2+ buffering and signalling. They are also major mediators of cell death. Toxic Ca 2+ accumulation in mitochondria is widely believed to initiate cell death in many cell types by opening the permeability transition pore (PTP). In non-neuronal cells, the PTP has been implicated as a Ca 2+ release mechanism in physiological Ca 2+ signalling. In neurons, Ca 2+ release from mitochondria has been attributed primarily to mitochondrial Na +/Ca 2+ exchange. Using fura-2 ratiometric microfluorimetry in acutely dissociated rat basal forebrain neurons, we show that mitochondria are able to buffer non-toxic Ca 2+ loads arising from caffeine-sensitive internal stores or from extracellular influx through voltage gated channels. We also show that these non-toxic Ca 2+ loads are reversibly released from mitochondria through the PTP and the Na +/Ca 2+ exchanger. Evoked Ca 2+ transients have characteristic peak and shoulder features mediated by mitochondrial buffering and release. Depolarizing mitochondria with carbonyl cyanide m-chlorophenylhydrazone (CCCP, 5 μM) causes release of mitochondrial Ca 2+ and prevents Ca 2+ uptake. In CCCP, the magnitudes of evoked Ca 2+ transients are increased, and the peak and shoulder features are eliminated. The PTP antagonist, cyclosporin A, (CSA, 2 μM) and the Na +/Ca 2+ exchange blocker, clonazepam, (CLO, 20 μM) reversibly inhibited both the shoulder features of evoked Ca 2+ transients and Ca 2+ transients associated with CCCP application. We suggest that central neuronal mitochondria buffer and release Ca 2+ through the PTP and Na +/Ca 2+ exchanger during physiological Ca 2+ signalling. We also suggest that CLO blocks both the PTP and the mitochondrial Na +/Ca 2+ exchanger.