Differential Ca²⁺ handling by isolated synaptic and non-synaptic mitochondria: roles of Ca²⁺ buffering and efflux

Mitochondria regulate intracellular calcium ion (Ca²⁺) signaling by a fine-tuned process of mitochondrial matrix (m) Ca²⁺ influx, mCa²⁺ buffering (sequestration) and mCa²⁺ release (Ca²⁺ efflux). This process is critically important in the neurosynaptic terminal, where there is a simultaneous high demand for ATP utilization, cytosolic (c) Ca²⁺ regulation, and maintenance of ionic gradients across the cell membrane. Brain synaptic and non-synaptic mitochondria display marked differences in Ca²⁺ retention capacity. We hypothesized that mitochondrial Ca²⁺ handling in these two mitochondrial populations is determined by the net effects of Ca²⁺ uptake, buffering or efflux with increasing CaCl₂ boluses. We found first that synaptic mitochondria have a more coupled respiration than non-synaptic mitochondria; this may correlate with the higher local energy demand in synapses to support neurotransmission. When both mitochondrial fractions were exposed to increasing mCa²⁺ loads we observed decreased mCa²⁺ sequestration in synaptic mitochondria as assessed by a significant increase in the steady-state free extra matrix Ca²⁺ (ss[Ca²⁺]_e) compared to non-synaptic mitochondria. Since, non-synaptic mitochondria displayed a significantly reduced ss[Ca²⁺]_e, this suggested a larger mCa²⁺ buffering capacity to maintain [Ca²⁺]_m with increasing mCa²⁺ loads. There were no differences in the magnitude of the transient depolarizations and repolarizations of the membrane potential (ΔΨ_m) and both fractions exhibited similar gradual depolarization of the baseline ΔΨ_m during additional CaCl₂ boluses. Adding the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE) inhibitor CGP37157 to the mitochondrial suspensions unmasked the mCa²⁺ sequestration and concomitantly lowered ss[Ca²⁺]_e in synaptic vs. non-synaptic mitochondria. Adding complex V inhibitor oligomycin plus ADP (OMN + ADP) bolstered the matrix Ca²⁺ buffering capacity in synaptic mitochondria, as did Cyclosporin A (CsA), in non-synaptic. Our results display distinct differences in regulation of the free [Ca²⁺]_m to prevent collapse of ΔΨ_m during mCa²⁺ overload in the two populations of mitochondria. Synaptic mitochondria appear to rely mainly on mCa²⁺ efflux via mNCE, while non-synaptic mitochondria rely mainly on P_i-dependent mCa²⁺ sequestration. The functional implications of differential mCa²⁺ handling at neuronal synapses may be adaptations to cope with the higher metabolic activity and larger mCa²⁺ transients at synaptosomes, reflecting a distinct role they play in brain function.