Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons

We sought to determine whether low-affinity, high-capacity mitochondrial Ca2+ uptake contributes to buffering physiological Ca2+ loads in sensory neurons. Intracellular free calcium concentration (Ca2+i) and intracellular free hydrogen ion concentration (H+i) were measured in single rat dorsal root ganglion (DRG) neurons grown in primary culture using indo-1 and carboxy-SNARF-based dual emission microfluorimetry. Field potential stimulation evoked action potential-mediated increases in Ca2+. Brief trains of action potentials elicited Ca2+i transients that recovered to basal levels by a single exponential process. Trains of > 25 action potentials elicited larger increases in Ca2+i, recovery from which consisted of three distinct phases. During a rapid initial phase Ca2+i decreased to a plateau level (450-550 nM). The plateau was followed by a slow return to basal Ca2+i Ca2+i transients elicited by 40-50 action potentials in the presence of the mitochondrial uncoupler carbonyl cyanide chlorophenyl hydrazone (CCCP), or the electron transport inhibitor antimycin A1, lacked the plateau, and the recovery to basal Ca2+i consisted of a single slow phase. Depolarization with 50 mM K+ produced a multiphasic Ca2+i transient and increased H+i from 74 +/- 3 to 107 +/- 8 nM. The rise in H+i was dependent upon extracellular Ca2+ and was inhibited by mitochondrial poisons. With mitochondrial Ca2+ buffering pharmacologically blocked, the recovery to basal Ca2+i was unaffected by removal of extracellular Na+. We conclude that large Ca2+ loads are initially buffered by fast mitochondrial sequestration that effectively uncouples electron transport from ATP synthesis, leading to an increase in H+i. Small Ca2+ loads are buffered by a nonmitochondrial, Na(+)-independent process.