A minimal presynaptic protein machinery mediating synchronous and asynchronous exocytosis and short-term plasticity

Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca ²⁺ influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca ²⁺ -triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca ²⁺ activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca ²⁺ -triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca ²⁺ concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca ²⁺ -evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.