Interactions Between SNAP-25 and Synaptotagmin-1 Are Involved in Vesicle Priming, Clamping Spontaneous and Stimulating Evoked Neurotransmission.

Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In the presence of PI(4,5)P2, vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction. SIGNIFICANCE STATEMENT: The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.

A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells.

Synaptotagmin-1 (Syt1) is the principal Ca(2+) sensor for vesicle fusion and is also essential for vesicle docking in chromaffin cells. Docking depends on interactions of the Syt1-C2B domain with the t-SNARE SNAP25/Syntaxin1 complex and/or plasma membrane phospholipids. Here, we investigated the role of the positively charged "bottom" region of the C2B domain, proposed to help crosslink membranes, in vesicle docking and secretion in mouse chromaffin cells and in cell-free assays. We expressed a double mutation shown previously to interfere with lipid mixing between proteoliposomes and with synaptic transmission, Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2 and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQ-induced docking was strictly PI(4,5)P2-dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca(2+)-triggered SUV-GUV fusion was restored by the Syt1 mutants. Finally, overexpressing the RQ-mutant in wild type cells produced no effect on either docking or secretion. We conclude that the positively charged bottom region in the C2B domain--and, by inference, Syt1-mediated membrane crosslinking--is required for triggering fusion, but not for docking. Secretory vesicles dock by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. The R398/399 mutations selectively disrupt the latter and hereby help to discriminate protein regions involved in different aspects of Syt1 function in docking and fusion. SIGNIFICANCE STATEMENT: This study provides new insights in how the two opposite sides of the C2B domain of Synaptotagmin-1 participate in secretory vesicle fusion, and in more upstream steps, especially vesicle docking. We show that the "bottom" surface of the C2B domain is required for triggering fusion, but not for docking. Synaptotagmin-1 promotes docking by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. Mutations in the C2B bottom surface (R398/399) selectively disrupt the latter. These mutations help to discriminate protein regions involved in different aspects of Synaptotagmin-1 function in docking and fusion.

Complexin arrests a pool of docked vesicles for fast Ca2+-dependent release.

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger--a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion.

Synaptic vesicles fuse with the plasma membrane in response to Ca(2+) influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca(2)(+)-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.

An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly.

Munc18-1, a SEC1/Munc18 protein and key regulatory protein in synaptic transmission, can either promote or inhibit SNARE complex assembly. Although the binary inhibitory interaction between Munc18-1 and closed syntaxin 1 is well described, the mechanism of how Munc18-1 stimulates membrane fusion remains elusive. Using a reconstituted assay that resolves vesicle docking, priming, clamping, and fusion during synaptic exocytosis, we show that helix 12 in domain 3a of Munc18-1 stimulates SNAREpin assembly and membrane fusion. A single point mutation (L348R) within helix 12 selectively abolishes VAMP2 binding and the stimulatory function of Munc18-1 in membrane fusion. In contrast, targeting a natural switch site (P335A) at the start of helix 12, which can result in an extended α-helical conformation, further accelerates lipid-mixing. Together with structural modeling, the data suggest that helix 12 provides a folding template for VAMP2, accelerating SNAREpin assembly and membrane fusion. Analogous SEC1/Munc18-SNARE interactions at other transport steps may provide a general mechanism to drive lipid bilayer merger. At the neuronal synapse, Munc18-1 may convert docked synaptic vesicles into a readily releasable pool.

SNAREpin assembly by Munc18-1 requires previous vesicle docking by synaptotagmin 1.

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.

Arrangements of proteins at reconstituted synaptic vesicle fusion sites depend on membrane separation.

Synaptic vesicle proteins, including N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Synaptotagmin-1 and Complexin, are responsible for controlling the synchronised fusion of synaptic vesicles with the presynaptic plasma membrane in response to elevated cytosolic calcium levels. A range of structures of SNAREs and their regulatory proteins have been elucidated, but the exact organisation of these proteins at synaptic junction membranes remains elusive. Here, we have used cryoelectron tomography to investigate the arrangement of synaptic proteins in an in vitro reconstituted fusion system. We found that the separation between vesicle and target membranes strongly correlates with the organisation of protein complexes at junctions. At larger membrane separations, protein complexes assume a 'clustered' distribution at the docking site, inducing a protrusion in the target membrane. As the membrane separation decreases, protein complexes become displaced radially outwards and assume a 'ring-like' arrangement. Our findings indicate that docked vesicles can possess a wide range of protein complex numbers and be heterogeneous in their protein arrangements.

Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25.

The neuronal protein complexin contains multiple domains that exert clamping and facilitatory functions to tune spontaneous and action potential-triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that, prior to fusion, the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitters in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

An Electrostatic Energy Barrier for SNARE-Dependent Spontaneous and Evoked Synaptic Transmission.

Information transfer across CNS synapses depends on the very low basal vesicle fusion rate and the ability to rapidly upregulate that rate upon Ca2+ influx. We show that local electrostatic repulsion participates in creating an energy barrier, which limits spontaneous synaptic transmission. The barrier amplitude is increased by negative charges and decreased by positive charges on the SNARE-complex surface. Strikingly, the effect of charges on the barrier is additive and this extends to evoked transmission, but with a shallower charge dependence. Action potential-driven synaptic release is equivalent to the abrupt addition of ∼35 positive charges to the fusion machine. Within an electrostatic model for triggering, the Ca2+ sensor synaptotagmin-1 contributes ∼18 charges by binding Ca2+, while also modulating the fusion barrier at rest. Thus, the energy barrier for synaptic vesicle fusion has a large electrostatic component, allowing synaptotagmin-1 to act as an electrostatic switch and modulator to trigger vesicle fusion.