Calcium Sensitive Ring-Like Oligomers of Synaptotagmin 1: Implications for Neurotransmitter Release

Abstract

The synaptic vesicle protein, Synaptotagmin-1 (Syt1) is required to couple Ca2+ influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Using negative stain electron microscopy, we find that the Syt1 assembles into ring-like oligomeric structures on lipid monolayers under physiological ionic strength and lipid composition in the absence of free Ca2+. These rings oligomers vary in diameter between 19 - 42 nm (with an average size of 30 ± 5 nm), corresponding to 12- 25 molecules of Syt1. The ring-like oligomers are sensitive to Ca2+ and are disrupted rapidly by the physiological concentrations of free Ca2+. Analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, the circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, we find that both Syt1 ring formation and its disruption by Ca2+ principally involve well-established functional surfaces of the C2B domain which are important for neurotransmission. This includes the Ca2+ -independent binding of the polybasic motif of C2B domain with phosphatidylinositol 4,5-bisphosphate (PIP2) as a prerequisite for rings to assemble. This suggests that ring formation may be triggered at a very early step in synaptic vesicle docking, as C2B-PIP2 interaction is required for docking in vivo. Ca2+ binding to the C2B domain and re-orientation/insertion into the membrane, both required for triggering synaptic transmission, disrupts the ring oligomers. We advance a simple and novel mechanism wherein Syt1 ring oligomer act as reversible washer/spacer to synchronize neurotransmitter release to Ca2+ influx. Supporting this hypothesis, we find mutations in Syt1 that specifically disrupt the ring oligomer formation results in dysregulation neuroexocytosis in PC12 cells.