Synaptic Vesicle Release Probability, Kinetics, and Ca-Sensitivity are Regulated by SNARE-Proteins

Abstract

Release of neurotransmitters (NTs) at neuronal synapses is accomplished by a multi-component machinery that senses calcium influx into the presynaptic terminal and responds by fusing the synaptic vesicle and plasma membranes and releasing NTs through a fusion pore. Decades of studies assumed that the vesicle release probability, the vesicle release rate and the Ca-sensitivity are regulated by the Ca-sensing component of the machinery. Here, we use molecular dynamics simulations of the multi-protein release machinery to demonstrate that the membrane fusion component of the machinery, consisting of the SNARE proteins, is equally important for regulation: when more SNARE complexes are assembled at the fusion site, the release probability and Ca-sensitivity increase, demonstrating a remarkable coupling between the Ca-sensing and membrane-fusing submachineries. Our simulations access the long timescales of NT release by implementing systematically coarse-grained representations of the neuronal SNARE complexes (Mostafavi et al., 2017) and the Ca-sensor for synchronous NT release, Synaptotagmin 1. The model quantitatively explains experiments in which NT release was enhanced at mouse Calyx of Held synapses by a constitutively open mutant of the t-SNARE Syntaxin, which increased the number of assembled SNARE complexes at the fusion site (Acuna et al., 2014): the vesicle release probability, NT release rate and apparent Ca-sensitivity of release were all increased, while the cooperativity of release was unaffected. We find the increased release probability and Ca-sensitivity are caused by acceleration of the final membrane-fusion step due to increased entropic forces that push the membranes together, and coupling of this final fusion step with the first stage of the process in which calcium triggers unclamping of the fusion machinery. Thus, the NT release machinery consists of membrane-fusing and Ca-sensing submachineries that are intimately coupled and both contribute to regulation of release.