Priming of synaptic vesicles to a readily-releasable state enables the fast speed of neurotransmitter release (< 1 ms) upon arrival of an action potential to a presynaptic terminal. The SNAREs syntaxin, SNAP-25 and synaptobrevin (yellow, green and red in the figures) form a tight four-helix bundle [1,2] that is partially assembled during priming. Fast, Ca2+-triggered membrane fusion is believed to be triggered when full SNARE complex assembly brings the vesicle plasma membranes together (see image above) and to involve a close functional interplay between the SNAREs, synaptotagmin (blue) and complexin (pink). However, the mechanism of membrane fusion and the nature of this interplay remain highly unclear.

Our research showed that the two C2 domains that form most of the cytoplasmic region of synaptotagmin (C2A and C2B) bind three and two Ca2+ ions (orange spheres) through loops at the tips of beta-sandwich structures [3-6]. The resulting change in electrostatic potential [7] induces phospholipid binding, and mutations that decrease or increase the apparent Ca2+ affinity of this activity cause parallel changes in the Ca2+ dependence of neurotransmitter release [8,9]. These findings demonstrated that synaptotagmin is the long-sought Ca2+ sensor that triggers release and that Ca2+-dependent phospholipid binding is key for synaptotagmin function.

We also showed that synaptotagmin function is tightly coupled to complexin [10], that complexin binds to the SNARE complex through a central helix that is extended into an accessory helix [11], and that this accessory helix inhibits neurotransmitter release [12], likely because of steric clashes with the vesicle that hinder membrane fusion [13,13] (panel A on the right). A crystal structure solved by the lab of Axel Brunger showed that synaptotagmin binds to the SNARE complex through the C2B domain [15]. This interaction allows simultaneous binding of the C2B domain to PIP2 on the plasma membrane (salmon spheres in panel A), which was supported by a cryo-EM structure [16], and of the SNARE complex to complexin. The resulting state is expected to inhibit membrane fusion because it hinders full zippering of the SNARE complex in the membrane-proximal region (panel A; note that the C-termini of synaptobrevin and syntaxin should be attached to the vesicle and plasma membranes, as indicated by the dashed lines, and hence the C-terminus of the SNARE cannot be assembled).

While it was thought for over 25 years that Ca2+ stimulates synaptotagmin-SNARE binding, we recently found that Ca2+ actually releases synaptotagmin-1 from membrane-anchored SNARE complexes to induce a tight, specific interaction with PIP2 and the membrane (panel B on the right) [17]. All these observations led us to propose a model whereby the primed state of synaptic vesicle includes an inhibited complex similar to that of panel A but with the four-helix bundle partially assembled. In this model, Ca2+ binding to synaptotagmin releases its inhibitory interaction with the SNAREs and allows cooperation of synaptotagmin with the SNAREs in membrane fusion, perhaps because of the ability of synaptotagmin to bridge two membranes and/or to induce membrane curvature [18].

We are currently devoting intense efforts to test this model by characterizing the putative inhibited complex formed by the SNAREs, synaptotagmin and complexin on a membrane, and investigating how the SNAREs and synaptotagmin induce membrane fusion. For this purpose, we are using a combination of NMR analyses on nanodiscs, cryo-EM studies on proteoliposomes, reconstitution assays and molecular dynamics simulations (see snapshot of the system in the image at the top).




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