Abstract
Mitochondria regulate intracellular calcium ion (Ca(2+)) signaling by a fine-tuned process of mitochondrial matrix (m) Ca(2+) influx, mCa(2+) buffering (sequestration) and mCa(2+) release (Ca(2+) efflux). This process is critically important in the neurosynaptic terminal, where there is a simultaneous high demand for ATP utilization, cytosolic (c) Ca(2+) regulation, and maintenance of ionic gradients across the cell membrane. Brain synaptic and non-synaptic mitochondria display marked differences in Ca(2+) retention capacity. We hypothesized that mitochondrial Ca(2+) handling in these two mitochondrial populations is determined by the net effects of Ca(2+) uptake, buffering or efflux with increasing CaCl(2) 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 mCa(2+) loads we observed decreased mCa(2+) sequestration in synaptic mitochondria as assessed by a significant increase in the steady-state free extra matrix Ca(2+) (ss[Ca(2+)](e)) compared to non-synaptic mitochondria. Since, non-synaptic mitochondria displayed a significantly reduced ss[Ca(2+)](e), this suggested a larger mCa(2+) buffering capacity to maintain [Ca(2+)](m) with increasing mCa(2+) 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 CaCl(2) boluses. Adding the mitochondrial Na(+)/Ca(2+) exchanger (mNCE) inhibitor CGP37157 to the mitochondrial suspensions unmasked the mCa(2+) sequestration and concomitantly lowered ss[Ca(2+)](e) in synaptic vs. non-synaptic mitochondria. Adding complex V inhibitor oligomycin plus ADP (OMN + ADP) bolstered the matrix Ca(2+) buffering capacity in synaptic mitochondria, as did Cyclosporin A (CsA), in non-synaptic. Our results display distinct differences in regulation of the free [Ca(2+)](m) to prevent collapse of ΔΨ(m) during mCa(2+) overload in the two populations of mitochondria. Synaptic mitochondria appear to rely mainly on mCa(2+) efflux via mNCE, while non-synaptic mitochondria rely mainly on P(i)-dependent mCa(2+) sequestration. The functional implications of differential mCa(2+) handling at neuronal synapses may be adaptations to cope with the higher metabolic activity and larger mCa(2+) transients at synaptosomes, reflecting a distinct role they play in brain function.