Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

Synchronous neurotransmitter release is a highly regulated process that results from the Ca 2+triggered fusion of synaptic vesicles with the presynaptic plasma membrane. This fusion process is driven by the assembly of the three neuronal SNAREs (soluble N-ethylmaleimide sensitive receptor proteins), syntaxin (Syx) and SNAP-25 in the plasma membrane and synaptobrevin (Syb) in the vesicle membrane (1, 2). The assembly of the SNAREs into a four helical bundle or SNARE complex is highly favorable and provides the energy required to overcome the energetic barriers to fusion. Several other proteins interact with the SNAREs and are critical to the proper assembly of the complex, including Munc18, Munc13 and complexin (3). The Ca 2+ -sensor for this process is the vesicle anchored protein, synaptotagmin 1 (Syt1), and the molecular event that initiates fusion is the binding of Ca 2+ to the two C2 domains of Syt1 (4). However, the mechanism by which Ca 2+ binding to the Syt1 C2 domains drives fusion is not understood.
The C2 domains of Syt1 are known to interact with and penetrate the membrane interface upon Ca 2+ binding (5,6). The second C2 (C2B) domain of Syt1 has a highly charged polybasic face that allows the domain to also associate in a Ca 2+ -independent manner to negatively charged membrane interfaces (7). This interaction is weak to monovalent lipid such as phosphatidylserine (PS) but much stronger to membrane interfaces containing PI (4,5)P2 (referred to here as PIP2) (8).
In the presence of Ca 2+ , the Ca 2+ binding loops and polybasic face work cooperatively to drive membrane association of the C2B domain. Synaptotagmin 1 is also known to associate with SNAREs, and several crystal structures have been generated showing the C2 domains of Syt1 in association with assembled SNAREs (9,10). However, the interactions are heterogeneous, and they are not observed in the presence of ATP under conditions that resemble those found within the cell (11). Synaptotagmin 1 may trigger fusion by altering the local lipid bilayer structure, thereby stimulating a conformational change in the nearby SNARE complex catalyzing membrane fusion (12). There is also evidence that Syt1 might alter the vesicle membrane/plasma membrane distance, and conceivably this event might trigger SNARE assembly (13,14). Synaptotagmin 1 also de-mixes PS upon membrane association (15), and this change in charged lipid distribution might modulate the membrane association of other proteins, such as Munc13 or the juxtamembrane regions of the SNAREs.
In addition to its charged polybasic face, the C2B domain of Syt1 has several highly conserved arginine residues in a region opposite the Ca 2+ -binding loops that we will refer to as the arginine apex. Mutating this arginine apex is reported to dramatically depress evoked excitatory postsynaptic currents in hippocampal neurons, and this apex is proposed to allow the C2B domain to bridge across bilayers (16). There is also direct evidence that the arginine apex associates with lipid bilayers and that it could facilitate contact of the C2B domain with two opposing bilayers (17). In the present work, we demonstrate that the arginine apex is associated with the membrane when the C2B domain is membrane bound and that membrane contact is dependent upon conserved arginine residues in the domain. The arginine apex also associates with the SNAREs in a manner consistent with crystal structures. However, unlike the membrane interaction, the interaction with the SNAREs is eliminated under conditions where fusion takes place. Using a single particle fusion assay with purified secretory granules, we find that the arginine apex does not affect the probability or kinetics of fusion but is necessary for the rapid release of secretory content, indicating that the apex has a role in controlling the expansion of the fusion pore. This result contrasts with that for the polybasic face, which alters the kinetics and probability of fusion but does not alter the rate of fusion pore expansion. The data indicate that there are two distinct molecular roles for Syt1 where different regions of the C2B domain make different contributions to the fusion process. We propose a novel mechanism for the action of Syt1 where the arginine apex of C2B helps position the domain at the negatively curved membrane surface that forms during the opening and expansion of the fusion pore. . The position of the polybasic face (red) and R398, 399 side chains are shown for C2B (blue). b) EPR spectra from an R1 label attached to site 173. In the presence of Ca 2+ , the domain inserts into the bilayer, which alters the sampling of rotamers by the R1 side chain and broadens the EPR spectrum. Figure 1 is a model for Syt1 where the two C2 domains are attached to the vesicle membrane through a long linker. In the present work, we utilize a soluble fragment of Syt1 containing the C2A and C2B domains (residues 136-421). Site-directed spin labeling may be used to both monitor membrane contact and measure membrane depth, and our approach involves the incorporation of the spin labeled side chain R1 into selected sites in the protein. As shown in Figure 1b, membrane insertion of a region of the domain, in this case the first Ca 2+ -binding loop of the C2A domain, produces a dramatic broadening in the EPR lineshape due to a reduction in the rotamers sampled by the R1 side chain.

The arginine apex makes interactions with the membrane interface when Syt1 C2AB is membrane bound. Shown in
To test for interactions in the arginine apex, spin labels were incorporated into three sites in the C2B domain that are depicted in Figure 2a, and the EPR spectra were recorded in the absence of membrane and in the presence of PC:PS vesicles with and without Ca 2+ . When the C2B domain is membrane associated in the presence of Ca 2+ , changes in the lineshapes from spin labels at sites 285, 349 and 350 are observed. The spectra show a slight broadening and a decrease in the residual hyperfine interaction, Azz', that may be due to the approach of the labels to the membrane interface. To confirm that these labels approach the membrane, progressive power saturation was used to provide an estimate of the distance of the labels to the membrane interface. As shown in Figure 2c, labels in the arginine apex lie close to the membrane interface in the presence of Ca 2+ and are about 2 showing conserved arginine residues in the apex, lysine residues in the polybasic face and the three sites to which R1 was attached. The arginine apex is on the opposite surface of the domain from two Ca 2+ -binding loops (CBL) that insert into bilayers in the presence of Ca 2+ . b) EPR spectra from the three labeled sites in the absence of membranes or the presence of PC:PS lipid vesicles. c) Membrane depth parameters obtained with and without Ca 2+ for labels near the apex.
Angstroms from a plane defined by the lipid phosphates on the aqueous side of the membrane (Table S1, supplement). These three labels also tend to be near the interface in the absence of Ca 2+ . This association may be facilitated by a weak association of the lysines forming the polybasic face of C2B (Figure 2a) to the PC:PS membrane interface. In the absence of Ca 2+ and under the conditions of this measurement, there is likely a significant concentration of aqueous C2B domain that is in equilibrium with a population of membrane associated C2B.
When PIP2 replaces PS in the membrane at equivalent charge densities, the EPR lineshapes and power saturation data also indicate that this region contacts the bilayer. As seen in Figure S1, site 350 makes a closer approach and site 285 is further away from the interface in PIP2 than in PS. Interestingly, site 349 does not show strong evidence for contact, which likely reflects the altered orientation for the C2B domain when bound to a PIP2 bilayer (8). The labeled position that shows the clearest evidence for membrane contact is site 350, and unlike the case for the interaction with PC:PS the interaction to PIP2 does not appear to be Ca 2+ -dependent. This likely reflects the enhanced Ca 2+ -independent membrane affinity of C2B to the PIP2 bilayer relative to the bilayer containing PS (8). Thus, we conclude that when C2AB is attached to the membrane interface, the arginine apex is also in contact with the bilayer surface.
Membrane contact at the apex is driven by conserved arginine residues. We then examined the effect of two sets of mutants, R398Q (RQ) and R398Q/R399Q (RQRQ) on the membrane depth Mutating the arginine apex reduces or eliminates membrane contact by the arginine apex. a) power saturation indicates that the RQ and RQRQ mutations reduce or eliminate membrane contact. b) EPR spectra obtained from site 285 for the pseudo wild-type (WT) and RQRQ mutant. EPR spectra are identical in the absence or presence of membranes for the RQRQ mutant. A depth parameter of 2 is obtained for an R1 label that lies approximately 4 Å from the lipid phosphate. When the labeled is further than 6 or 7 Å from the phosphates, the depth parameter becomes independent of label position. parameters from the two most closely associating positions, 285 and 350, on the C2B domain of Syt1. Figure 3a shows the results of power saturation measurements for the wild-type C2AB versus the single RQ and double RQRQ mutations (Table S2, supplement). Each arginine mutation shifts the apex further towards the aqueous phase or eliminates the membrane interaction, with the double RQRQ mutation showing the largest effects. For example, in the presence of PC:PS the RQRQ mutation shifts the position of 285R1 approximately 5 Å towards the aqueous phase. In PC:PIP2, the RQRQ mutation eliminates the membrane interaction of 285R1. Shown in Figure 3b are comparisons of the EPR spectra from site 285 with and without membrane for the wild-type C2AB and RQRQ mutation. The spectra obtained from site 285 in the presence of the RQRQ mutation are identical with and without membrane indicating that there is no membrane contact at this site in the presence of the RQRQ mutation.
The arginine apex contacts membrane reconstituted SNARE proteins. The arginine apex has been observed to interact with the soluble SNARE complex by x-ray crystallography (9). We also examined the EPR spectra from the three spin labeled sites at the apex of C2B to test for this interaction with SNARE complexes embedded in lipid bilayers. Shown in Figure 4a are EPR spectra from sites 285, 349 and 350 in the presence of PC:PS membranes with or without membrane reconstituted SNAREs composed of full-length Syx, SNAP-25, and soluble Syb. The spectra for sites 285 and 350 have reduced normalized amplitudes and broadened hyperfine extrema (Azz') indicating that the motion and rotamer sampling of the nitroxide at these sites is being sterically restrained by interactions with the SNAREs. We examined the rotamers available at these sites as predicted from the crystal structure (PBD ID: 5CCH) using the program MMM (18) and the result is shown in Figure 4b. According to this model, the R1 label at all three sites should contact the SNAREs, with sites 350 and 285 having the greatest reduction in the available rotamers and site 349, which projects away from the SNAREs, having the least. This is consistent with the data in Figure 4a and indicates that these EPR data in the presence of membrane reconstituted SNAREs is consistent with the model from crystallography.

The addition of ATP or the presence of PIP 2 in the membrane eliminates the SNARE interactions but does not eliminate the membrane interactions of the arginine apex.
Next, we tested the effect of the RQ mutations and mutations in SNAP-25 on the association of Syt1 with the SNAREs. Interactions between Syt1C2AB and the SNAREs are likely driven by charge interactions, and we mutated three negatively charged residues D51, E52 and E55 to alanine in the N-terminal segment of SNAP-25. As seen in Figure 4c, a comparison of the EPR spectra from spin labeled sites in the Syt1C2AB arginine apex show no differences between PC:PS membranes alone and PC:PS membranes containing SNAREs with this AAA mutant. The result indicates that the AAA mutation eliminates the Syt1C2AB-SNARE interaction. Importantly, as shown below, this mutation somewhat reduces docking of secretory vesicles and their overall fusion, but it does not alter the Ca 2+ dependent release mode (fusion pore opening) and Ca 2+ stimulated fusion increase. Figure 4d compares normalized amplitudes of spectra obtained from 285R1 and 350R1 in the presence of membrane reconstituted SNAREs, membrane reconstituted SNAREs having the AAA mutation, as well as membrane reconstituted SNAREs in the presence of the RQ and RQRQ mutations. Both single and double RQ mutations, as well as the AAA mutation, yield normalized amplitudes like those obtained for membranes lacking the SNAREs (composed only of PC:PS), indicating that the Syt1/SNARE interaction no longer takes place. The addition of 1 mM ATP/Mg 2+ (a typical cytoplasmic concentration) also eliminates interactions between C2AB and the membrane reconstituted SNAREs as does the presence of 5 mol% PIP2 in the membrane (Figure 4d). . The arginine apex contacts membrane reconstituted SNAREs, but ATP or PIP 2 in the bilayer, eliminate the interaction. a) EPR spectra in the absence (blue trace) or presence (red trace) of membrane reconstituted SNARE complex composed of full-length Syx, SNAP-25, and soluble Syb. The broadened components of the hyperfine tensor (arrows) indicate tertiary contact of the R1 side chain with SNAREs. b) Model (PDB ID 5CCH) with available rotamers for the R1 side chains at the three sites. c) Comparison of EPR spectra with and without membrane reconstituted SNAREs containing the AAA mutation in SNAP-25. d) Normalized amplitudes for EPR spectra from sites 285R1 and 350R1. Larger amplitudes indicate loss of tertiary contact with the SNAREs.
As seen in Figures 3 and 4, both the membrane and SNARE interactions of the arginine apex are sensitive to the RQ and RQRQ mutations. Thus, both interactions require these conserved arginine residues and are likely driven by electrostatics. However, the SNARE interaction is eliminated by PIP2, whereas the membrane interaction is not. SNARE interactions are also eliminated by ATP, which is also not the case for the membrane interaction (see Figure S2). Figures 3 and 4 indicate that conserved arginine residues in the apex of the C2B domain facilitate membrane contact of the apex under the conditions where fusion takes place. To better define their role in the fusion process, we used a single particle fusion assay to examine fusion between chromaffin secretory granules purified from PC12 cells (also referred to as dense core vesicles, DCVs) and reconstituted planar supported membranes containing recombinant syntaxin-1a and dSNAP-25 (dodecylated SNAP-25). These purified chromaffin granules were previously shown to be composed of the same molecular machinery as synaptic vesicles purified from rat brain (19). An important feature of this assay when investigating the machinery of secretion is the ease of engineering stable knockdowns into PC12 cells and thus producing cell-derived vesicles with syt1 depletions and no other changes.

Mutations in the arginine apex do not alter fusion probability but alter the rate of fusion pore opening. The data in
The chromaffin granules are labeled by expressing the releasable content marker neuropeptide Y (NPY)-mRuby, and their binding to planar supported membranes in a SNARE dependent manner (schematic in Figure 5a, left) can be observed using total internal reflection fluorescence microcopy (TIRFM) (20). Granule binding is accompanied by an increase in fluorescence within the TIRF field, where a subset of the granules fuse after a variable time delay (Fig. 5b). Fusion is marked by a decrease in fluorescence followed by a rapid spike in fluorescence signal and then a further decay in signal ( Fig. 5b and c). The initial decay in the signal is caused by the loss of NPY-mRuby from the granule as it diffuses into the cleft between the supported membrane and its supporting slide during an initial slow release phase. The rapid increase in fluorescence is a result of the rapid collapse of the granule as fluorescent marker is rapidly driven into the evanescent field of the TIRFM. As NPY-mRuby continues to diffuse away from the fusion site, the signal decays. The origin of this signal has been previously described in detail (19,20) and is modulated by the lipid geometries that stabilize or destabilize fusion pores (21).
When granules are depleted of endogenous synaptotagmin isoforms, Ca 2+ -dependent fusion is lost; however, addition of the soluble C2AB domain recovers the Ca 2+ dependence (Fig. 5d). The intensity of the spike in fluorescence (ΔIC) is indicative of the amount of content released from the granule during the fast phase of the fusion event after the initial slow release. The value of ΔIC reflects the mode of contents release, or the timing of the fusion event, and in our model it is dependent upon the speed with which the granules transition between the initial slow and fast phases of contents release. As seen in Figure 5c, this release mode is modulated by the presence of C2AB and 100 µM Ca 2+ when granules depleted of endogenous synaptotagmin are used. The C2AB domain was previously shown to catalyze a structural transition in the SNARE complex by interactions mediated through the membrane bilayer (12). This structural transition involves a change in SNARE orientation relative to the membrane, i.e. a transition from a trans-mimicking conformation to a cis-mimicking conformation, which can be monitored using site directed fluorescence interference contrast microscopy (sdFLIC) (Figure 5a, right) (12). As seen in Figure  5e this conformational change is dependent on C2AB and calcium. Combining the TIRF fusion and sdFLIC assays creates a powerful tool to examine secretory granule binding, fusion probability, content release, SNARE complex conformational changes, and kinetics of fusion, all under the same conditions. In the context of the various Syt1 mutations and their modulated interactions with membranes and SNAREs, this approach allows us to tease out the role of Syt1 in fusion. Figure 5f shows the effects of three sets of mutants in C2B on granule binding, fusion efficiency, content release, structural changes in the SNAREs, and kinetics of fusion. Here, we examined a single arginine mutation in the apex (RQ), mutation of both arginines in the apex (RQRQ), or lysine mutations in the polybasic face (KAKA). In the absence of Ca 2+ , granule binding is mediated by SNAREs, and in the presence of Ca 2+ it is mediated by both SNAREs and the vesicleresident calcium binding protein CAPS (20). Figure 5f shows that these mutations in C2B have no effect on granule binding. When the percentage of granule fusion events is examined, only mutations in the polybasic face (KAKA) depress the fusion efficiency, while mutations in the arginine apex have no effect. However, when we examine the timing of the fusion event (the release mode), the KAKA mutant has no effect while mutations in the arginine apex strongly affect the release mode. Thus, mutations in the apex strongly affect the transition from the slow to fast phase of content release, i.e. the transition from the opening to the dilation of the fusion pore. In contrast, as shown previously (12), mutations in the arginine apex produce only small perturbations in the conformation change of SNARE complex (measured by sdFLIC) compared to wild-type C2AB, while stronger effects on this orientational change are observed for the KAKA mutation. The polybasic face also had a greater effect on the kinetics of the delay time from granule binding to fusion, while the arginine apex mutations had only a slight effect.
As seen in Figure 5f, the SNARE structural changes strongly correlate with the fusion efficiency and kinetics, and it is interesting to note that the effects of these C2B mutants track with their effect on C2AB membrane affinity. The KAKA mutant, which had the largest effect on SNARE structural changes and fusion, is known to reduce the membrane affinity of C2AB in the presence of PIP2 (8). In contrast, the membrane affinity of C2AB was measured for the RQRQ mutation in the apex and was found to produce no significant change in affinity (see Figure S3). The data in Figure 5f indicate that the membrane binding of Syt1 C2AB is necessary to initiate fusion, but that the membrane interactions made by the arginine apex are required to for the proper timing of the fusion event and the rapid opening of the fusion pore.  Figure 2 indicate that the arginine apex contacts negatively charged membranes whenever the C2B domain is membrane associated. However, other regions of C2B also make membrane contact. In the presence of PIP2 and under the same conditions that attach the arginine apex, the Ca 2+ -binding loops penetrate and the polybasic face interacts with PIP2 (8). These interactions are evident in the EPR spectra shown in Figure 6a, where the EPR spectra and power saturation data show that these regions of C2B are interacting with the membrane interface and with PIP2 (Figure 6b and Table S3).

The C2B domain makes simultaneous membrane contact at the arginine apex, the polybasic face and the Ca 2+ -binding loops. The data shown in
Inspection of the spectra from sites 304 and 329 indicates that for virtually the entire population of C2B, these regions are membrane associated or interacting with PIP2. Multiple components in the spectra that would indicate an equilibrium between aqueous and membrane associated C2B are not seen. Such an equilibrium would be more difficult to distinguish in the spectra from the apex (Figure 2), where spectra corresponding to bound and unbound apex are similar. Nonetheless, the presence of a mixture of membrane associated and aqueous states is not obvious. Thus, a large fraction of C2B must undergo simultaneous membrane contact at these sites. Shown in Figure 6c are the allowable rotamers of the nitroxide spin label at each of the membrane interacting sites. It is not possible to simultaneously satisfy these interactions with a flat planar bilayer interface or two parallel bilayer surfaces; however, these interactions can be satisfied if the C2B domain interacts with a curved membrane interface.

Removal of PIP 2 alters granule binding, fusion, fusion pore opening, and SNARE conformational changes.
The association of the polybasic face with the membrane interface seen in Figure 6a is driven by PIP2, and we tested the effect of PIP2 on granule fusion and SNARE conformation as described above for the C2B mutants. As shown in Figure 6d, removal of PIP2 in the presence of 100 μM Ca 2+ reduces granule binding, fusion efficiency, slows the fusion timing or release mode, alters SNARE conformation, and reduces fusion kinetics. The loss of PIP2 will reduce C2AB affinity and should have a similar effect as the KAKA mutant. But the loss of PIP2 also produces an effect on the opening of the fusion pore that is similar to that of the RQ and RQRQ mutants (Figure 5f). The change in calcium stimulated granule binding mediated by PIP2 was previously demonstrated to be dependent on CAPS which is a membrane associated calcium binding protein with a PIP2 specific PH domain (20).
The SNAP-25 AAA mutation has no effect on granule contents release and the opening of the fusion pore. As shown above in Figures 4c, d, mutation of SNAP-25 to replace three negatively charged residues (the SNAP-25 AAA mutation) eliminated the SNARE binding of the arginine apex. We tested to see what effect these mutations would have on the granule docking and fusion and the opening of the fusion pore. As seen in Figure 6e, in the presence of Ca 2+ the AAA mutation reduces, but does not eliminate, granule binding and fusion efficiency. However, the effects of the AAA mutation are also seen in the absence of Ca 2+ or C2AB, indicating that they are not mediated by C2AB. In contrast to the RQ and RQRQ mutations (Figure 5f), the AAA mutation has no effect on the release mode or timing of the fusion event. Since this mutation eliminates the SNARE interactions made by the C2B arginine apex, SNARE interactions of the apex are not necessary to mediate changes in Ca 2+ -dependent fusion pore opening. Figure 6. The C2B domains makes simultaneous membrane contact at multiple sites that cannot be explained by docking to a planar bilayer surface. a) EPR spectra in an aqueous state compared with spectra in the presence of PC:PIP 2 bilayers in the presence of EGTA or Ca 2+ . b) Membrane depth parameters indicate that site 173 inserts into the PIP 2 bilayer in the presence of Ca 2+ . Sites on C2B including 304, 323 and 329 also contact with bilayer, with or without Ca 2+ . c) Allowable rotamers for labeled sites that contact the membrane interface including two near the arginine apex. The membrane contact seen at these sites could be accommodated by a curved membrane surface. This would place a PIP 2 headgroup in a binding pocket at the polybasic face. d) The effect of PI(4,5)P 2 on granule binding, fusion, release profile, SNARE orientation, and fusion kinetics in the presence of 100 µM Ca 2+ (membrane composition was 25:25:15:30 bPC:bPE:bPS:Chol and either 5% PI or 4% PI with 1% PI(4,5)P 2 ). e) The effect of the AAA mutation in SNAP-25 on granule binding, fusion, release profile, SNARE orientation, and fusion kinetics in the presence of 100 µM Ca 2+ (membrane composition 32:32:15:20:1 bPC:bPE:bPS:Chol:PI(4,5)P 2 ).

Discussion
Despite its central importance for synchronous neurotransmitter release, the molecular mechanisms underlying Syt1 function are presently unclear. Molecular models for Syt1 in association with SNAREs have been generated by X-ray crystallography, and they suggest that the Ca 2+ regulatory event involves a control of the SNAREs by a direct interaction with Syt1 (9, 10). However, other data indicate that the interactions of Syt1 with SNAREs are weak, heterogeneous, and are of secondary importance to the interactions made by Syt1 to membranes (11).
In the present work, we examined a region on the C2B domain that lies opposite the Ca 2+ -binding loops that contains a pair of conserved arginine residues at sites 398 and 399. Mutating this pair of residues to glutamine (RQRQ mutation) is known to have a significant effect on synchronous neurotransmitter release and excitatory postsynaptic potentials (16), and our initial goal was to determine whether the effect of these mutations was acting at the level of a membrane or direct SNARE interaction. As shown in Figures 2 and S1, the arginine apex associates with membranes whenever Syt1 C2AB is membrane associated. The apex also associates with membrane reconstituted SNAREs in a manner that is consistent with molecular structures generated by crystallography, and both membrane and SNARE interactions are weakened or eliminated by the RQ and RQRQ mutations. However, the data presented here indicate that only the membrane interactions of the C2B apex occur under conditions where fusion takes place.
The interaction made by the arginine apex with membrane reconstituted SNAREs is eliminated when PIP2 is present in the membrane (Figure 4d), or when ionic conditions mimic those expected within the cell. In addition, mutation of three basic residues within SNAP25 (the AAA mutation) also eliminates the Syt1-SNARE interaction (Figures 4c, d). However, the AAA mutation does not abolish membrane docking or fusion (Figure 6e), and it has no effect on the rate of fusion pore expansion during the fusion event, indicating that interactions between the C2B arginine apex and SNAREs are not involved in Ca 2+ -triggered neuropeptide release. In contrast, when PIP2 is present in the membrane, the apex of C2B contacts membranes under all conditions we examined and is progressively weakened by the RQ and RQRQ mutations. These mutations in the apex have a profound effect on the rate at which the fusion pore opens and the concentration of vesicle content that is released quickly into the TIRF field (Figure 5f). These data demonstrate that the membrane interactions of the apex of Syt1, and not the SNARE interactions, regulate the fusion pore expansion.
Previous work has shown that Syt1 C2AB binds preferentially to membranes rather than SNAREs when PIP2 is present, an interaction that is mediated by the polybasic face of C2B (8,11). Here, the KAKA mutation in the polybasic face of C2B did not alter the opening of the fusion pore (Figure 5f), rather it altered SNARE conformational changes and fusion probability and kinetics. The result indicates that the apex plays a role in Syt1 function that is distinct from that of the polybasic face and that there are at least two molecular roles played by C2B that are important for fusion.
Isoforms of synaptotagmin are known to have different behaviors with respect to Ca 2+ -sensitivity and synchronous release (22). The arginine apex appears to be important in controlling the release mode of synaptotagmins, as shown by a comparison of the behavior of the Syt1 RQ and RQRQ mutants with that of synaptotagmin-7 (Syt7). Using the same single particle fusion assay as that used here, Syt7 demonstrates a substantially lower release rate than does WT Syt1 (23); however, the rates of release for Syt7 are similar to those seen here for either the RQ or RQRQ mutations of Syt1 (Figure 5f). A comparison of the sequence of the arginine apex for Syt1 and Syt7 shows that Syt7 is RQ rather than RR, and thus is identical in this regard to the Syt1 RQ mutant. We speculate that Syt7 and the RQ mutant of Syt1 look similar in terms of the rate of fusion pore opening because both proteins have the same residues in this apex.
How might membrane interactions of the apex of C2B act to mediate membrane fusion and alter the characteristics of the fusion pore? In previous work, membrane interactions made both by the arginine apex and the Ca 2+ -binding loops on opposite surfaces of the domain suggested a model where C2B bridged across two parallel bilayer planes, perhaps ultimately functioning to shorten the vesicle-plasma membrane distance in the presence of Ca 2+ (14,17). However, the data presented here indicate that in the presence of PIP2, the polybasic face also contacts the PIP2 headgroup (Figure 6a,b), so that three regions of the C2B domain make membrane contact. The EPR lineshapes indicate that the interaction of the polybasic face and the Ca 2+ -binding loops are stable interactions that are not transient. This is more difficult to determine for the arginine apex, but nonetheless simultaneous membrane interactions of each of these three regions must occur for at least some period. It is not possible to dock the C2B domain to either one planar bilayer surface or to two parallel planar bilayer surfaces so that contact in all three regions is satisfied. But simultaneous interactions in all three regions of C2B can be satisfied if the domain is interacting with a curved bilayer interface (Figure 6c). Indeed, recent simulations suggest that mixtures of lipids likely to mediate fusion can form punctate surfaces with regions of negative curvature (24), which might accommodate this type membrane interaction of C2B. Figure 7 is a model for the membrane interactions made by Syt1 that incorporates what is known about the molecular interactions made by C2B and the effect of mutations in the arginine apex on granule binding and fusion. In this model, Syt1 is interacting at the negatively curved surface that is present following the formation of a hemifusion intermediate. As the fusion pore forms, the C2B domain of Syt1 interacts with this negatively curved surface making contact at the Ca 2+ -binding loops, arginine apex and polybasic face. In doing so, the C2B domain of Syt1 helps mediate the transition during a fusion event to a fully open pore. It might mediate this transition by either lowering an energy barrier for pore expansion or by preventing the system from getting trapped at an early intermediate state during the pore opening (25). By weakening or eliminating membrane contact at the apex with either the RQ or RQRQ mutations, the C2B domain no longer assumes the correct orientation across the cytoplasmic side of the fusion pore, and the mode of contents release or progression to a fully open fusion pore is slowed (Figure 5f). Other than the RQ and RQRQ mutations, PIP2 is the other component that is seen to strongly influence the release mode or pore opening when it is removed (Figure 6e). Interestingly, the polybasic face is aligned facing into a region of high negative curvature in this model. Since it interacts with PIP2, this lipid would be driven into an interface of negative curvature. PIP2 is a lipid that alone will tend to form micelles (26,27), and its position within the negative curve of the growing fusion pore would not be energetically favored. Conceivably, the presence of PIP2 in this region of the pore, driven by strong electrostatic interactions with the polybasic face of C2B, might act to destabilize an initial state in the fusion pore and drive it to a fully open state. In (a) after docking to the bilayer and in the presence of ATP/Mg 2+ (orange) the SNARE complex, syntaxin (red), SNAP-25 (green), synaptobrevin (purple), and Syt1 C2B (blue), promote partial lipid mixing by either zippering or contacting plasma membrane PIP 2 at the arginine apex (orange) and the polybasic face (red). Upon calcium influx (green), Syt1 C2A inserts into the synaptic vesicle membrane, PS, and C2B reorients, binding the Ca 2+ -binding loops at a 3rd membrane contact point. The C2B domain of Syt1 might position at the site of fusion as shown in (b). This orientation would sequester PIP 2 into the strained membrane stalk, further destabilizing the membrane and promoting pore opening.

Shown in
In summary, the data presented here are consistent with a novel model for the role of Syt1 where the C2B domain interacts with the negatively curved membrane surface during the initial stages of fusion to mediate expansion of the fusion pore. Using both EPR spectroscopy and single particle fusion experiments we demonstrate that the arginine apex of C2B helps control the opening or dilation of the fusion pore and mediate the transition between the initial pore opening and a fully open fusion pore. We demonstrate that the C2B domain interacts both with membranes and membrane associated SNAREs; however, only the membrane interaction takes place under the conditions where fusion occurs. As a result, the expansion of the fusion pore is mediated by a membrane interaction of the C2B arginine apex. Mutations that lie in the polybasic face of C2B alter the fusion probability but have no effect on the expansion of the fusion pore once fusion occurs, indicating that there are at least two molecular roles for Syt1 in triggering fusion.