Calcium Sensitive Ring-Like Oligomers of Synaptotagmin: Implications for Regulation of Neurotransmitter Release

Abstract

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Using electron microscopy we find an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium. Rings vary in diameter from 18 nm to 43 nm, corresponding to 11 to 26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and liposomal bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that the C2B interacts with the membrane and is involved in ring formation, while the C2A domain points radially outwards. SYT rings are rapidly disrupted by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: the ring acts as a spacer to prevent the completion of SNARE complex assembly thereby clamping fusion (in the absence of calcium). When the ring disassembles in the presence of calcium, then fusion proceeds unimpeded.