Calcium dependence of neurotransmitter release at a high fidelity synapse

The Ca²⁺-dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles' cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca²⁺-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca²⁺ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca²⁺ sensor for vesicle priming. Ca²⁺ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca²⁺ concentration up to 50 μM. The rate of vesicle replenishment during sustained elevated intracellular Ca²⁺ concentration exhibited little Ca²⁺-dependence. Finally, quantitative mechanistic release schemes with five Ca²⁺ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca²⁺ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.