(627a) Neurotransmitter Release Relies on Coupled Calcium Sensing and Membrane Fusion

Synaptic transmission is a highly synchronized process, coordinated by a complex protein machinery that responds to a stimulus by releasing neurotransmitters (NTs) that bind postsynaptic receptors. On arrival of an action potential at the axon terminal that triggers opening of voltage-gated calcium channels, the machinery senses the influx of calcium and fuses the neuronal plasma membrane and synaptic vesicle membrane to release NTs through a fusion pore. Thus, the NT release machinery performs two key functions – it acts as a Ca-sensitive clamp, preventing premature fusion in the absence of a stimulus, and triggers vesicle fusion on sub-millisecond timescales in response to elevated calcium. The membrane fusion machinery is highly conserved across diverse cell types, enabling the regulation of various exocytotic events, including NT release and insulin secretion. Gaining a comprehensive understanding of synaptic transmission is crucial in the investigation of neurodegenerative diseases and synaptopathies, but despite the extensive characterization of synaptic transmission by electrophysiological techniques, understanding the molecular mechanisms that drive NT release remains a major challenge in the field. In a long-standing view, Ca-triggered unclamping and membrane fusion are distinct tasks, carried out by different machinery components: the Ca-sensor Synaptotagmin (Syt) and the SNARE fusion proteins. Our simulation results challenge this notion. We found there is no clear division of labor between Syt and the SNAREs, and that the NT release machinery is instead highly cooperative, whereby unclamping and fusion are closely intertwined.

Simulating the collective behavior of large protein machineries with many components is a challenge in the field of modeling and computation. Here we developed a molecularly detailed mathematical model of a minimal NT release machinery that accounts for Ca-triggered unclamping and membrane fusion. To access the millisecond timescales of NT release, we employed highly coarse-grained representations, while maintaining crucial biophysical properties. The sensitivity of the NT release machinery to the level of [Ca] has been measured from Ca uncaging experiments. The relationship between the rate of synaptic release and the Ca concentration was shown to follow a power law with an exponent of 3-5. This result has been conventionally interpreted as a requirement for 3-5 Ca ions to sequentially bind to Syt to trigger fusion. However, at low [Ca], the measured release rates are inconsistent with this model. While our model reproduces the power law dependence of release rates on [Ca], our simulations revealed that this cooperativity is an emergent property of the NT release machinery, unrelated to the number of Ca ions required for fusion. Surprisingly, we also found that the likelihood of the machinery activating release after the arrival of an action potential is determined not only by Ca-triggered unclamping, but also by machinery components associated with membrane fusion. Additionally, the brief delay between the influx of Ca into the presynaptic terminal and the first fusion events is mainly dependent on the unclamping rate rather than the fusion rate. A mechanism emerges by which vesicles can only fuse during a finite time window following an action potential, when [Ca] is elevated in the presynaptic terminal and the SNAREs are unclamped. With more unclamped SNAREs, the fusion rates are higher, so that more NT release occurs during this short time window. Together, these results suggest that the Ca-triggered unclamping and membrane fusion functions of the NT release machinery are closely coupled, and their coordination is critical for efficient synaptic transmission.