Molecular mechanisms of neurotransmitter release

Nerve cells communicate by releasing the contents of neurotransmitter-bearing synaptic vesicles into the space between adjoining cells. This process depends on a handful of proteins that promote vesicle and nerve cell membrane fusion. We use high-resolution structural methods and biophysical tools to capture this machinery at different stages of vesicle fusion. These structures then provide the framework for further investigations, primarily using electron microscopy, into the functional and dynamic aspects of the system.

SNARE proteins, found in both nerve cell and vesicle membranes, set the stage for fusion by zipping together into a parallel, four-helix bundle that juxtaposes the two membranes. We determined the first x-ray crystal structure of the neuronal SNARE complex, as well as the structures of other key components of the synaptic release machinery. We visualized the SNARE complex bound to the Ca2+-sensor synaptotagmin-1 and to the regulator complexin, revealing two interfaces. The “primary” interface between synaptotagmin and the SNARE complex is conserved by primary sequence, has been observed in several independent crystal structures, and it has been validated by several subsequent experiments. Follow-up studies of the “tripartite” interface are in progress. The structures of these synaptic complexes suggest that they represent primed and locked states. Action-potential-driven Ca2+ ions bind to the synaptotagmin proteins, unlock the complexes, and trigger membrane fusion on a sub-millisecond timescale.

After fusion has occurred, SNARE complexes are recycled by the ATPase NSF, which breaks down the SNARE complex into its individual components. We visualized this molecular machine at near-atomic level and obtained the first glimpses of how this SNARE-recycling machine works. The SNARE complex resembles a rope with a left-handed twist, and NSF uses adapter proteins called SNAPs to grasp the “rope” in multiple places. The SNAPs wrap around the SNARE complex with a right-handed twist, suggesting that the disassembly occurs via a simple unwinding motion that frees the zipped SNARE proteins.

We are also using structural and functional studies to explore other machinery relevant to neurotransmitter release, such as factors involved in priming and pre-synaptic plasticity.  This research may one day provide new possibilities for targeting therapeutics to control neurotransmitter release.

A schematic diagram of the synaptic vesicle fusion cycle. Also shown are structures of the neuronal SNARE complex (Sutton et al., Nature 395, 347-353, 1998); the SNARE/Cpx1/Syt1 complex (Zhou et al., Nature 548, 420-525, 2017); and the SNARE/αSNAP/NSF complex (White et al. eLife, e38888, 1662018).