How Many Synaptotagmin Molecules does it Take to Clamp a SNARE Complex?

Abstract

Neurotransmitter (NT) release is achieved by a specialized protein machinery that senses presynaptic calcium entry following an action potential, fusing vesicle and plasma membranes and releasing vesicle contents through the resultant fusion pore. Synaptotagmin 1 (Syt) is the Ca sensor for synchronous NT release, and is thought to clamp SNARE-mediated fusion at basal [Ca], but to release the clamp at elevated [Ca]. Synaptic vesicles host ∼15-20 Syt molecules and ∼70 copies of the SNARE protein VAMP. However, the number of SNARE complexes driving fusion is controversial, and the number of Syt molecules clamping the SNAREs is unknown. Importantly, two key experimental studies in which the number of SNARE complexes per vesicle was modulated showed more SNAREs lead to greater and faster NT release (Acuna et al., 2014; Arancillo et al., 2013). Here, we present coarse-grained molecular dynamics simulations of NT release, varying the number of assembled SNARE complexes and Syt molecules. In Ca-uncaging simulations, the apparent cooperativity of release was proportional to the stoichiometric ratio of Syt molecules to SNARE complexes, with a ratio of 2:1 reproducing the well-established 4th power scaling of post-synaptic current amplitude with [Ca]. Turning to action potential-evoked release, increasing the number of 2:1 Syt-SNARE modules increased the release probability and the Ca-sensitivity of release, recapitulating the experimental findings of Acuna et al. and Arancillo et al. In our sumulations Syt molecules were initially assembled in ring-like oligomers (Wang et al., 2014), to which SNARE complexes docked via the primary interface (Zhou et al., 2015). Elevation of [Ca] triggered ring disassembly and unclamping, allowing SNAREs to assemble into a ring. Entropic forces drove ring expansion, leading to fusion. Our results suggest that additional SNAREs potentiate NT release by enhancing the entropic forces driving fusion.