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⁺](i). 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 A 1, 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.