Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

6 dual inhibitory and stimulatory roles of the membrane bridging activity of Munc13-1 are also consistent with the effects of point mutations that disrupt interactions between the various Munc13-1 domains , with the finding that a H567K mutation that unfolds the C1 domain of Munc13-1 impairs vesicle priming but increases the vesicular release probability (Basu et al., 2007;Rhee et al., 2002), and with the observation that deletion of the C1 or C2B domain of unc-13 enhances neurotransmitter release in C. elegans, yet deletion of both domains strongly hinders release (Michelassi et al., 2017). Nevertheless, the validity of this two state model and the functional importance of the polybasic face formed by the C1 and C2B domains remain to be demonstrated.
The study presented here was designed to test this model and investigate the functional consequences of mutations in predicted membrane-binding residues of the Munc13-1 C1-C2B region using a combination of molecular dynamics (MD) simulations, electrophysiological experiments in neurons and biochemical and reconstitution assays in vitro. We find that K603E or R769E single point mutations in the C1C2B polybasic face of Munc13-1 disrupt Ca 2+ -independent liposome binding and bridging, as well as stimulation of liposome fusion in reconstitution assays, and severely impair synaptic vesicle priming in mice autaptic cultures. Conversely, a K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca 2+ -binding loops, which have milder effects on liposome binding, bridging and fusion, do not affect vesicle priming; however, Ca 2+ -evoked release and the release probability are enhanced by the K720E mutation and impaired by K706E. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. These results strongly support the notion that binding of the C1-C2B region of Munc13-1 to the plasma membrane is critical for synaptic vesicle priming and that a switch from a perpendicular to a slanted orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

Models of Munc13-1 C1-C2B-membrane interactions in the absence and presence of Ca 2+
The notion that the C1-C2B region of Munc13-1 can bind to membranes via different surfaces in the absence and presence of Ca 2+ emerged from analysis of the distribution of charged residues in these surfaces   (Fig. 1B,C). To derive models of these putative membrane-binding modes, we performed MD simulations using a model of a fragment spanning the C1, C2B and MUN domains of Munc13-1 (C1C2BMUN) based on its crystal structure , and a square membrane with a lipid composition that mimics that of the plasma membrane (Chan et al., 2012). In one simulation, we placed the Ca 2+ -free C1C2BMUN model above the cytoplasmic leaflet in an approximately perpendicular orientation with the polybasic face close to the membrane, and ran a simulation of 100 ns. The orientation of C1C2BMUN became even more perpendicular in the first 10 ns and during the remaining 90 ns oscillated around an almost completely perpendicular orientation such as that observed at the end of the simulation We carried a second simulation in which we included two Ca 2+ ions bound to the corresponding binding sites of the C2B domain (Shin et al., 2010) and C1C2BMUN was placed in a more slanted orientation such that the DAG-and Ca 2+ /PIP2-binding sites of the C1 and C2B domains, respectively, were close to the membrane. A range of slanted orientations of C1C2BMUN were visited during the 86 ns simulation, oscillating during the last 70 ns around the orientation observed in the last pose, which was even more slanted than the initial orientation (  (Shin et al., 2010) inserted partially into the membrane through its hydrophobic side 8 chains while its basic residues interacted with the phospholipid head groups. It is noteworthy that this helix also interacts with the membrane in the perpendicular orientation adopted in the Ca 2+ -free simulation, which allows a more extensive membrane-interacting surface involving the sides of the C1 and C2B domains, as well as the linker between them (Figure 1-Figure supplements 1B and 2A). In the slanted, Ca 2+ -bound orientation, the interaction surface is smaller because it involves only the tips of the C1 and C2B domain, but hydrophobic groups of the C2B domain helix and the C1 domain insert into the membrane, and a PIP2 head group located between the Ca 2+ -binding loops is close to the Ca 2+ ions (Figure 1-Figure supplements 1D and 2B). Since phospholipid head groups are known to complete the coordination spheres of Ca 2+ ions bound to C2 domains, dramatically increasing their Ca 2+ affinity (Verdaguer et al., 1999;Zhang et al., 1998), it seems likely that phospholipid-Ca 2+ coordination and the insertion of hydrophobic groups into the membrane provide the driving force for this binding mode and a Ca 2+ -induced change in orientation of Munc13-1 with respect to the membrane.
We would like to emphasize that these simulations were biased by the initial orientations and a much more thorough analysis will be required to explore the possible binding modes of the Munc13-1 C1-C2B region with the plasma membrane. However, these short simulations were dynamically very stable around the two distinct basins, with and without Ca 2+ , supporting the notion that Munc13-1 can adopt two dramatically different orientations with respect to the plasma membrane. The simulations yielded chemically reasonable binding modes and help to visualize how changes in the orientation of Munc13-1 can drastically alter the distance between a vesicle and the plasma membrane (Fig. 1D,E). Note also that, although we postulate that Ca 2+ favors the switch from the perpendicular to the slanted orientation, both binding modes are likely possible in the absence and presence of Ca 2+ , and other factors such a more complete assembly of the SNARE complex may induce the switch to the slanted orientation even in the absence of Ca 2+ . The observed binding modes provide useful frameworks for the design of mutations to disrupt Munc13-1-membrane interactions involving the C1C2B region. Using these models, we designed 9 the following mutations to investigate the functional importance of the distinct faces of the C1-C2B region and test the hypothesis that this region mediates binding of Munc13-1 to membranes in two different orientations that underlie in part its role in regulating neurotransmitter release: i) K603E and K720E mutations in the C1 and C2B domain, respectively, probed residues in the polybasic face that are far from the Ca 2+ -binding sites of the C2B domain and are predicted to interact with the lipids in the Ca 2+independent binding mode ( . Note that a mutation of this residue to Trp in Munc13-2 led to a gain-of-function, increasing the Ca 2+ sensitivity of neurotransmitter release (Shin et al., 2010).

Differential disruption of neurotransmitter release by mutations in the C1C2B region of Munc13-1
To investigate the functional consequences of the K603E, K706E, K720E and R769E mutations in the C1C2B region of Munc13-1, we analyzed synaptic responses in autaptic hippocampal cultures from Munc13-1/2 double knockout (DKO) mice rescued with Munc13-1 carrying these point mutations individually. All We first analyzed their impact on SV priming as measured by depletion of the readilyrelease pool (RRP) of vesicles induced by hypertonic solution (Rosenmund and Stevens, 1996). We found that DKO neurons rescued with the Munc13-1 bearing the K603E mutation in the C1 domain or the R769E mutation in one of the C2B domain Ca 2+ -binding loops exhibited a drastically reduced RRP ( Fig. 2A).
However, no significant impact on RRP size was observed for the K720E mutation in the C2B domain, despite the proximity of K720 to K603 (Fig. 1B), or for the K706E mutation in another C2B domain Ca 2+binding loop ( Fig. 2A).
We next examined the influence of the four mutations on Ca 2+ -evoked neurotransmitter release.
The vesicular release probability (Pvr), the likelihood that an action potential causes the fusion of a primed and fusion competent vesicle, can be readily calculated by dividing the excitatory postsynaptic current (EPSC) and sucrose evoked charges (Reim et al., 2001;Rosenmund and Stevens, 1996). The K603E and R769E mutations decreased Ca 2+ -evoked release but did not alter the Pvr significantly (Fig. 2B,C). In contrast, the K706E mutation decreased both the EPSC amplitude and the Pvr, while the K720E mutation increased both evoked release and the Pvr (Fig. 2B,C). These effects on the efficiency of release observed for the K706E and K720E mutants were further confirmed by recordings with a pair pulse protocol consisting of two consecutive AP-induced EPSCs with an inter-stimulus interval of 25 ms (Fig. 2D). Thus, the K603E and R769E mutations did not alter the paired-pulsed ratio (PPR), but the K706E mutant exhibited an increased PPR, which is typical of synapses with low Pvr, whereas we observed a decreased PPR for the K720E mutant, as expected from its increased Pvr. We also assessed the impact of these mutations on spontaneous release by analyzing the frequency of miniature postsynaptic currents (mEPSCs). We observed a decrease in mEPSC frequency for K603E and R769E, the two mutants that exhibited impaired priming, whereas the K706E mutation did not affect spontaneous release and K720E, the mutant with enhanced Pvr, had increased mEPSC frequency ( Overall, these results show that two basic residues in the C1C2B region of Munc13-1, K603 and R769, play important roles in SV priming, whereas two other basic residues, K706 and K720, modulate the vesicular release probability and have opposite effects. To gain further insights into the roles of these residues, we analyzed neurotransmitter release in rescue experiments with Munc13-1 double mutants that combined the two point mutations in residues that are far from the C2B domain Ca 2+ -binding sites (K603E/K720E) or the two mutations in the C2B domain Ca 2+ -binding loops (K706E/R769E), as well as a quadruple mutant where the four basic residues were replaced (K603E/K720E/K706E/R769E). The K603E/K720E mutant exhibited impaired sucrose-induced, Ca 2+ -evoked and spontaneous release, without significant changes in Pvr or PPR (Fig. 3 , and appeared to have a tendency to lower Pvr than wild type (WT), but the difference did not reach statistical significance (Fig. 3C). Correspondingly, the PPR observed for the K706E/R769E mutant showed a tendency to facilitation that was not statistically significant. Finally, we observed that the K603E/K720E/K706E/R769E quadruple mutant exhibited the lowest number of responsive cells and that these were strongly deficient in priming, spontaneous release and, particularly, Ca 2+ -evoked release, while the Pvr and RRP observed for this mutant had similar non-statistically significant tendencies as the K706E/R769E mutant ( show that the mutations that impair priming, K603E and R769E, dominate the phenotypes of these double and quadruple mutants and the effects of the mutations that specifically affect evoked release, K706E and K720E, are at least partially masked by these dominant effects. Overall, these data show that mutations in the C1C2B region of Munc13-1 can have effects on both SV priming and Ca 2+ -evoked release, and can lead to both loss-of-function and gain-of-function. These findings support the notion that the C1C2B region is involved in Ca 2+ -independent interactions through the polybasic face that are critical for priming and in additional interactions involving in part the C2B domain Ca 2+ -binding loops that affect the probability of evoked release, consistent with the two state model of to what appear to be opposite effects (loss-or gain-of-function) suggests that release is modulated by a delicate balance between inhibitory and stimulatory interactions involving the C1C2B region, which is also a key aspect of the model. It seems likely that at least some of the interactions involving the C1C2B polybasic face that mediate the initial bridging of the vesicle and plasma membranes by Munc13-1, leading to primed vesicles, need to be released for Munc13-1 to adopt the more slanted orientation that facilitates SNARE complex zippering and membrane fusion. The interplay between these two events may underlie the distinct effects of the K603E and K720E mutations, as both mutations can impair the initial bridging but facilitate the transition to the slanted orientation to different degrees (see discussion).

Distinct effects of mutations in the C1C2B region of Munc13-1 on short-term plasticity
We next investigated how the mutations in basic residues of the C1C2B region affect synaptic responses during repetitive stimulation by applying 10 Hz stimulus trains on autaptic cultures of Munc13-1 DKO neurons rescued with WT and comparing them side-by-side with rescues by the Munc13-1 mutants.
Analysis of these data is complicated by the fact that the observed changes in EPSC amplitudes during repetitive stimulation can be caused by various mechanisms, including depletion of the RRP, alterations in the efficiency of replenishment of the RRP and changes in the release probability. To help distinguishing between these mechanisms, we prepared plots of normalized responses, which inform on differences in the extent of depression or facilitation during the stimulus trains, and plots of absolute EPSC amplitudes, which can help to clarify the mechanisms related to use and re-fill of primed synaptic vesicles (Shin et al., 2010) (Fig. 4).
As expected , DKO neurons rescued with WT Munc13-1 displayed substantial depression in the beginning of the stimulus train that can be attributed to RRP depletion. The R769E single mutant exhibited stronger depression than WT Munc13-1 (Fig. 4L), which likely arises at least in part from impairment in the kinetics of RRP replenishment, as this mutant had a strong defect in priming 13 but no deficit in Pvr (Fig. 2). However, the K603E mutant also had impaired priming and yet it exhibited similar depression to WT Munc13-1 in the beginning of the train, with a tendency to have higher EPSC amplitudes than WT as the train progressed (Fig. 4C). These findings suggest that the fusogenicity of primed vesicles is altered during the stimulus train and is consistent with the notion that, as intracellular Ca 2+ accumulates during repetitive stimulation, Ca 2+ -binding to the Munc13-1 C2B domain favors more slanted orientations of Munc13-1 (Fig. 1E) that mediate release more efficiently than the perpendicular orientations. Thus, based on this model, K603 contributes to binding of the C1C2B region to the plasma membrane in the orientations present in the absence of Ca 2+ that mediate initial priming, but not in the slanted orientations favored upon Ca 2+ binding to the C2B domain that are increasingly populated and lead to enhanced fusogenicity of the primed vesicles during the stimulus train ( The K720E mutant displayed stronger depression than WT, based on normalized EPSC plots ( Fig.   4F), but plots of absolute EPSC amplitudes show that the K720E mutant starts with higher amplitudes that depress faster over time, reaching the same steady state as WT (Fig. 4E). Thus, the stronger depression observed initially for the K720E mutant is most likely due to the initially higher release probability exhibited by this mutant (Fig. 2C) but, based on the two-state model of Fig. 1D,E, the K720E mutation does not affect release later during the stimulus training because K720 does not participate in binding in the slanted orientations favored by Ca 2+ accumulation. In contrast, the K706E mutation led to depression that was similar to that observed for WT Munc13-1 in normalized EPSC plots (Fig. 4I), and absolute EPSC amplitudes show that the EPSCs observed for the K706E mutation were smaller than those of WT throughout the stimulus train (Fig. 4H). Since the Pvr of the K706E mutant was lower than that of WT ( Fig.   2C), these observations suggest that the release probability remained low for the K706E mutant throughout the stimulus train. The effects observed for the K706E mutation are opposite to those caused 14 by a mutation in the corresponding lysine residue of Munc13-2 to tryptophan (Shin et al., 2010) and may arise because the mutation hinders the transition to the slanted orientation, but they may also reflect a decreased fusogenicity of primed vesicles. Finally, the effects observed for the K603E/K720E and (K706E/R769E) double mutants ( Fig. 5A-F) and the K603E/K720E/K706E/R769E quadruple mutants ( Fig.   5G-I) appeared to be a combination of the effects caused by the single mutations, with some dominance by the K603E and the R769E mutations.
At synapses, phorbol esters increase the release probability at least in part by activating Munc13-1 through its C1 domain (Basu et al., 2007;Rhee et al., 2002), mimicking the effects of DAG. The two state model predicts that this potentiation arises because DAG/phorbol esters favor the slanted orientations of Munc13-1 that facilitate SNARE complex formation and fusion. To test this notion, we analyzed the effects of the mutations in basic residues of the Munc13-1 C1C2B region on potentiation by a phorbol ester [phorbol 13,]. PDBu caused a robust potentiation of EPSCs in DKO autaptic cultures rescued with WT Munc13-1 (Fig. 6A,B), as expected, and the K603E and R769E mutants exhibited even higher potentiation (Fig. 6A). These results suggest that PDBu partially compensates for the priming defects caused by the K603E and R769E mutations. In contrast, the K720E mutant displayed less potentiation than WT Munc13-1, which, based on the two state model, may arise because the K720E mutation already facilitates the transition from the perpendicular to the slanted orientation and thus has an intrinsically higher release probability. The potentiation observed for the K706E mutant was similar to that observed for WT Munc13-1, suggesting that this mutation does not affect the transition between orientations but rather downstream events that lead to synaptic vesicle fusion. The K603E/K720E and K706E/R769E double mutants exhibited similar enhancements of PDBu-induced potentiation as the K603E and R769E single mutants, respectively, showing again that these mutations dominate the phenotypes of the double mutants. The results obtained for the rescue with the K603E/K720E/K706E/R769 quadruple mutant need to be interpreted with caution because this mutant exhibited a high-number of non-15 responsive neurons and that the EPSCs for the responsive neurons were very weak. However, it is noteworthy that we still were able to observe PDBu potentiation for this mutant.
Overall, these results show that the basic residues in the Munc13-1 C1-C2B region modulate the potentiation of synaptic responses by PDBu and, together with the data obtained with repetitive stimulation, they support the notion proposed in the two state model that two faces of the C1-C2B region are critical for Munc13-1-dependent short-term plasticity.

Mutations in the Munc13-1 C1-C2B polybasic region impair membrane binding
The proposal that C1C2BMUNC2C can bridge membranes in two different orientations (Fig. 1D,E) emerged from the structural studies of the C1C2BMUN fragment that revealed the polybasic face of the C1-C2B region , as well as from liposome clustering assays and reconstitution experiments with liposomes containing synaptobrevin (V-liposomes) and liposomes containing syntaxin-1 and SNAP-25 (Tliposomes). Thus, fusion of these liposomes in the presence of Munc18-1, NSF and αSNAP requires Ca 2+ and the Munc13-1 C1C2BMUNC2C fragment, and depends on the ability of C1C2BMUNC2C to bridge the liposome membranes, but the liposome clustering activity of C1C2BMUNC2C is comparable in the absence and presence of Ca 2+ , indicating that Ca 2+ induces a switch to an active orientation required for fusion (Liu et al., 2016;Quade et al., 2019). Mutagenesis studies demonstrated the key importance of the C2C domain for the ability of C1C2BMUNC2C to bridge membranes and support liposome fusion (Quade et al., 2019), but the role of the C1C2B region has not been tested. To address this question and attempt to rationalize the functional effects of the basic mutations in the C1C2B region observed with electrophysiology, we used a combination of assays that measure liposome binding, clustering or fusion.
We first tested the effects of these mutations on binding to liposomes with the lipid composition that we normally used for T-liposomes, which mimics that of the plasma membrane (Ma et al., 2013), using liposome co-sedimentation assays. All mutations decreased Ca 2+ -independent liposome binding to some degree compared to wild type (WT) C1C2BMUNC2C (top panels of Fig. 7A,B), which may arise in part because they all decrease the overall positive electrostatic potential in the region. Among the single point mutations, K603E and R769E impaired binding most strongly, while K720E had an intermediate effect and K706E exhibited the weakest effect on binding. Ca 2+ -independent liposome binding was strongly disrupted by the two double mutations and the quadruple mutation. These results are consistent with the Ca 2+independent binding mode predicted for the C1-C2B region ( Moreover, the strong impairment of Ca 2+ -independent liposome binding caused by the K603E and R769E mutations correlate with the disruption of priming caused by these mutations ( Fig. 2A), supporting the notion that Ca 2+ -independent membrane binding of the C1C2B region is important for SV priming.
Ca 2+ -dependent liposome binding was not substantially decreased by any of the single mutations or by the K603E/K720E double mutation. The K706E/R769E mutation appeared to impair Ca 2+ -dependent liposome binding to a moderate degree, while binding was almost abrogated by the quadruple mutation ( Fig. 7A, middle panel, and 7B, lower panel). These results suggest that Ca 2+ substantially increases the affinity of C1C2BMUNC2C for the liposomes. Such an increase may not be observable for WT C1C2BMUNC2C because under the conditions of our experiments binding is likely strong enough for saturation in the absence and presence of Ca 2+ , but Ca 2+ -induced enhancement of binding becomes detectable when the mutations decrease the overall affinity. For the same reason, the effects of single mutations on Ca 2+dependent liposome binding may not be detectable even if the mutated residues contribute to binding, but the effects observed for the double mutants support the notion that K706 and R769 contribute more to Ca 2+ -dependent binding than K603 and K720, consistent with the model of Fig. 1C, Figure 1-Figure supplement 2B. The strong effect caused by the quadruple mutation indicates that the latter residues also contribute to Ca 2+ -dependent liposome binding, likely by decreasing the overall electrostatic potential in the region.
We next analyzed the ability of the various mutants to cluster protein-free liposomes with the same lipid compositions of V-and T-liposomes using dynamic light scattering (DLS). Figure  but note that these averaged radii need to be interpreted with caution because of the difficulty in accurately calculating populations of very large particles. As observed previously (Liu et al., 2016), WT C1C2BMUNC2C caused strong liposome clustering, and the amount of clustering was comparable in the absence and presence of Ca 2+ (Fig. 8A). In the absence of Ca 2+ , the liposome clustering activity was severely impaired by the single K603E and R769E mutations, the two double mutations and the quadruple mutation, whereas the single K706E and K720E mutants still supported robust liposome clustering ( Fig.   8B-H). In contrast, all the mutants except the quadruple mutant induced substantial liposome clustering in the presence of Ca 2+ . These results generally correlate with the liposome binding data, and reveal that the liposome clustering activity is affected more strongly by the mutations in the absence than in the presence of Ca 2+ .
Overall, these data show that the C1-C2B region is critical for the liposome bridging activity of Munc13-1 C1C2BMUNC2C and support the notion that distinct membrane binding modes of the C1-C2B region mediate membrane bridging in the absence and presence of Ca 2+ , as predicted from the models of

Mutations in the Munc13-1 C1-C2B polybasic region impair liposome fusion
We next turned to our reconstitution assays in which fusion between V-and T-liposomes in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C, NSF and αSNAP is assessed by simultaneously monitoring lipid and content mixing (Liu et al., 2016). The strict Ca 2+ dependence of fusion in these assays arises because of Ca 2+ binding to the Munc13-1 C2B domain. The two state model postulates that such binding favors a more slanted orientation of C1C2BMUNC2C, accelerating trans-SNARE complex formation and fusion, and that this phenomenon underlies at least in part the facilitation of neurotransmitter release due to accumulation of intracellular Ca 2+ during repetitive stimulation (Shin et al., 2010). To test whether the Ca 2+ concentration required to activate the Munc13-1 C2B domain in the liposome fusion assays is consistent with this proposal, we monitored lipid mixing between V-and T-liposomes in the presence of WT Munc18-1, Munc13-1 C1C2BMUNC2C, NSF and αSNAP as a function of Ca 2+ concentration. The fluorescence of the Ca 2+ -sensing dye Fluo-4 was used to measure the Ca 2+ concentration in each point of the titration, and lipid mixing between the liposomes was monitored simultaneously from the de-quenching of Did-lipids present in the V-liposomes. We observed a steep dependence of lipid mixing on the Ca 2+ concentration ( Fig. 9A), and fitting plots of the lipid mixing observed at 1500 or 700 s as a function of Ca 2+ concentration to a Hill equation yielded EC50s of 584 nM and 966 nM, respectively (Fig. 9B). These data show that the Munc13-1 C2B domain is activated in these fusion assays at submicromolar Ca 2+ concentrations, comparable to those expected to accumulate near Ca 2+ channels in a presynaptic active zone during repetitive stimulation.
We then examined the effects of the mutations in basic residues of the C1-C2B region of Munc13-1 on liposome fusion using these assays, in which the V-and T-liposomes commonly have synaptobrevinto-lipid ratio 1:500 and syntaxin-1-to-lipid ratio 1:800 (Liu et al., 2016). Ca 2+ -dependent liposome fusion was not impaired by any of the single point mutations or by the double K603E/K720E mutation ( Fig.   10A,B,E). However, the double K706E/R769E mutation did impair fusion strongly, and fusion was abrogated by the quadruple K603E/K720E/K706E/R769E mutation (Fig. 10A,B,E). Thus, K706 and R769, which are in the Ca 2+ -binding loops of the C2B domain, are critical for Ca 2+ -dependent activation of fusion, while K603 and K720 did not have detectable inhibitory effects. However, it is important to note that two aspects of these assays limit their application to analyze the effects of mutations on fusion. First, inhibitory effects of the mutations on Ca 2+ -independent fusion cannot be analyzed because there is no fusion in the absence of Ca 2+ in these assays. Second, because fusion with WT C1C2BMUNC2C is so efficient upon addition of Ca 2+ , gain-of-function effects cannot be observed, and moderate inhibitory effects may also be masked.
To analyze the effects of the mutations on Ca 2+ -independent fusion, we performed analogous assays but using Munc18-1 bearing a gain-of-function mutation (D326K) that supports some Ca 2+independent liposome fusion (Sitarska et al., 2017) (Fig. 10C,D). The Ca 2+ -independent component of fusion, as assessed from the extent of content mixing, was somewhat inhibited by the K706E mutation, was impaired more strongly by the K720E mutation, and was abolished by all other mutations (Fig. 10D).
Since lipid mixing was disrupted to a smaller extent by the mutations and hence yielded a higher dynamic range for quantification, we measured the lipid mixing at 300 s, right before Ca 2+ addition, in repeat experiments performed under the same conditions (Fig. 10F). This analysis showed that lipid mixing was strongly impaired by all the mutations except the K706E and K720E mutations, which inhibited lipid mixing moderately. These results correlate with those obtained in the analyses of SV priming ( Fig. 2A, 3A) and Ca 2+ -independent liposome clustering ( Fig. 8), confirming the importance of the membrane bridging activity of C1C2BMUNC2C for its ability to mediate liposome fusion and supporting the notion that this activity is critical for SV priming.
To address the second issue and assess whether inhibitory or stimulatory effects of the mutations on Ca 2+ -dependent fusion in the experiments of Fig. 10A,B might not have been observed because of its high efficiency with WT C1C2BMUNC2C, we performed additional assays under conditions that we recently developed to render Ca 2+ -dependent fusion less efficient and highly sensitive to the Ca 2+ sensor synaptotagmin-1 (Syt1) (Stepien and Rizo, 2021). In these assays, we monitored fusion between liposomes containing synaptobrevin and Syt1 at 1:10,000 and 1:1,000 protein-to-lipid (P/L) ratios, respectively, with liposomes containing syntaxin-1 and membrane-anchored SNAP-25 at 1:5,000 and 1:800 P/L ratios, respectively. The results that we obtained were similar to those observed under our standard conditions variability that we normally observe in these assays (Stepien and Rizo, 2021).
Overall, the results obtained in the liposome fusion assays strongly support the proposal that binding of the C1-C2B region of C1C2BMUNC2C to the T-liposomes is crucial for liposome clustering and fusion, and that different binding modes involving this region in the absence and presence of Ca 2+ underlie the drastic activation of liposome fusion observed in our assays. Moreover, our data suggest that these assays recapitulate at least to some extent the effects of the mutations on neurotransmitter release in neurons, as the K603E and R769E mutations that disrupt synaptic vesicle priming ( Fig. 2A) exhibit the strongest disruption of Ca 2+ -independent fusion among the single mutations ( Fig. 10B,C,F), and the impairment of Ca 2+ -dependent fusion by the double K706E/R769E mutation but not the double K603E/K720E mutation (Fig. 10A,B,E) correlates with the effects of these mutations on the vesicular release probability (Fig. 3C).

Discussion
Research for over three decades has led to major advances in our understanding of the basic mechanisms underlying the priming and fusion of synaptic vesicles to release neurotransmitters, including the notions that Munc18-1 and Munc13-1 coordinate assembly of SNARE complexes in an NSF-αSNAP-resistant manner (Ma et al., 2013;Prinslow et al., 2019) and that this pathway enables the multiple modes of regulation of release probability during presynaptic plasticity that depend on Munc13-1 (Park et al., 2017;Sitarska et al., 2017;Stepien et al., 2019). This large multiple domain protein plays fundamental roles in this process by accelerating opening of the syntaxin-1 closed conformation (Ma et al., 2011;Yang et al., 2015) and bridging the vesicle and plasma membranes (Liu et al., 2016;Quade et al., 2019). Thus, it seems likely that Munc13-1-dependent regulation of neurotransmitter release involves at least in part alteration of these activities. Indeed, the finding that the C1C2B region has two putative faces that can bind to membranes led to an intriguing model postulating that membrane bridging by Munc13-1 in two orientations modulates neurotransmitter release . Here we have tested this model and examined the role of the C1C2B region in neurotransmitter release. We find that basic residues in this region play key roles in SV priming, in dictating the vesicular release probability and in modulating shortterm plasticity. The differential effects of mutations in these basic residues on neurotransmitter release are mirrored in part in their effects in liposome clustering and fusion assays. Overall, these results support the notion that changes in the orientation of Munc13-1 with respect to the membranes are critical for neurotransmitter release and some forms of short-term presynaptic plasticity.
The notion that the highly conserved C-terminal region of Munc13-1 containing the C1, C2B, MUN and C2C domains bridges the vesicles and plasma membranes arose naturally from the discovery of its 22 rod-like architecture, with membrane binding domains on opposite ends of the highly elongated MUN domain (Liu et al., 2016;Xu et al., 2017). At the same time, the structural studies revealed that the Munc13-1 C1C2B region contains a large polybasic face adjacent to the region containing the DAG/phorbol ester-binding site of the C1 domain and the Ca 2+ /PIP2-binding region of the C2B domain (Fig. 1B,C), suggesting that Munc13-1 can bind to the plasma membrane through two different faces that yield different orientations . This prediction is supported by our MD simulations, which indicate that membrane interactions mediated by the polybasic face involve multiple ionic interactions in an extensive surface and lead to an almost perpendicular orientation of the Munc13-1 rod with respect to the membrane. In contrast, the simulations suggest that interactions mediated by the DAG/Ca 2+ /PIP2binding face involve a smaller area and are favored by Ca 2+ -phospholipid coordination and insertion of hydrophobic residues into the membrane, in addition to ionic interactions involving basic residues, leading to a very slanted orientation (Fig. 1D,E, Figure 1- Figure supplements 1 and 2). These observations support the notion that the C1C2B region binds to membranes through the larger polybasic face in the absence of Ca 2+ , adopting perpendicular orientations, whereas slanted orientations are favored by Ca 2+ and DAG (PIP2 might also favor this orientation but also participates in interactions with the polybasic face).
It is important to realize that the orientation of Munc13-1 is likely dynamic, particularly in the presence of other forces. For instance, the perpendicular orientation of Munc13-1 is expected to facilitate initiation of SNARE core complex assembly but prevent full assembly, resulting in a tug-of-war between Munc13-1 and the SNAREs in the absence of Ca 2+ that is dramatically tilted toward full SNARE complex assembly and fusion when Ca 2+ binding to the C2B domain favors slanted orientations. However, the balance can also be tilted towards slanted orientations in the absence of Ca 2+ by the energy associated with further assembly of the SNARE complex and by proteins that bind to the SNARE complex such as Syt1 and complexins (see below). Indeed, a large amount of physiological data can be explained with a model postulating that primed vesicles exist in a dynamic equilibrium between a loosely primed state (LS), in 23 which SNARE complexes are partially assembled and Munc13-1 bridges the vesicle and plasma membrane in a perpendicular orientation, and a tightly primed state (TS) where the SNARE complex is more fully assembled and Munc13-1 bridges the membranes in a slanted orientation (Neher and Brose, 2018). In this scenario, vesicles in the TS state are much more likely to be released by an action potential than those in the LS state, and accumulation of Ca 2+ during repetitive stimulation increases the vesicular release probability because it shifts the equilibrium toward the TS state. Note however that methods used to measure the RRP such as application of sucrose likely release vesicles in both the LS and TS states. This two state model and the proposed nature of the two membrane-binding modes of the C1-C2B region underlying the two states are strongly supported by our data.
The strong impairments of liposome binding and clustering in the absence of Ca 2+ caused by the K603E and R769E mutations in Munc13-1 C1C2BMUNC2C (Fig. 7, 8) show that the polybasic face is indeed critical for Ca 2+ -independent membrane binding and membrane bridging. These mutations also caused the strongest impairments of Ca 2+ -independent liposome fusion among the single mutants (10C,D,F). The finding that the K720E mutation in the polybasic face has smaller effects on Ca 2+ -independent liposome binding, clustering and fusion suggests that K720 has a smaller energetic contribution to membrane binding than those of K603 and R769. The K706E mutation exhibited the smallest inhibitory effects on liposome binding and fusion in the absence of Ca 2+ , which correlates with the fact that K706 is not in the polybasic face.
Ca 2+ enhanced the affinity of all the mutants for liposomes (Fig. 7) and increased the clustering activity of the mutants that had the weakest clustering ability (K603E, R769E, and the two double mutants), except the quadruple mutant (Fig. 8). Hence, although we did not observe Ca 2+ -induced increases in liposome binding and clustering for WT C1C2BMUNC2C because its liposome affinity and clustering activity are very high, Ca 2+ did increase binding and clustering when these activities were impaired by mutation. These increases may arise in part because Ca 2+ binding to the C2B domain enhances the overall positive electrostatic potential and in part because the Ca 2+ -dependent binding mode with slanted orientations occurs with a higher affinity than the Ca 2+ -independent binding mode involving the polybasic face. The four single mutants and the K603E/K720E double mutant supported Ca 2+ -dependent fusion with comparable efficiency as WT C1C2BMUNC2C (Fig. 10A,B,E) even though the Ca 2+ -dependent clustering activity of all these mutants was somewhat lower than that of WT (Fig. 8), suggesting that fusion efficiency can remain very high as long as the clustering activity is above a certain threshold. Interestingly, the double K706E/R769E mutation in the C2B domain Ca 2+ -binding sites disrupted Ca 2+ -dependent liposome fusion severely (Fig. 10A,B,E) even though its clustering activity is comparable to that of C1C2BMUNC2C with the K603E/K720E double mutation in the polybasic face (Fig. 8). This finding strongly supports the notion that a Ca 2+ -induced switch to a slanted orientation is critical for Ca 2+ -induced enhancement of liposome fusion.
Our electrophysiological studies support the biological relevance of at least some of the results obtained in our in vitro assays and reveal additional effects of the mutations that could not be discerned with the liposome fusion assays, providing critical evidence in support of the two state model. The observation that only the K603E and R769E mutants among the single Munc13-1 mutants exhibited severely impaired SV priming ( Fig. 2A) clearly correlates with the much stronger disruption of Ca 2+independent liposome binding, clustering and fusion caused by these mutations, compared to the K706E and K720E single mutants (Fig. 7, 8, 10C,D,F). These results provide compelling evidence that the polybasic face of the Munc13-1 C1C2B region plays a critical function in synaptic vesicle priming. Together with the key importance of the C2C domain for membrane bridging and SV priming (Quade et al., 2019), these data strongly support the notion that this critical function involves bridging of the vesicle and plasma membranes by respective interactions involving the C2C domain and the C1C2B region of Munc13-1. The K603E and R769E mutants also exhibited similar impairments in Ca 2+ -evoked release that arose because of the defects in priming, as the Pvr remained analogous to that of WT Munc13-1 (Fig. 2B-C), and the 25 potentiation of release by PDBu was somewhat larger for both mutants than for WT Munc13-1 (Fig. 5A).
These observations suggest that binding of PDBu to the C1 domain can partially compensate for the defects in priming caused by these two mutations by favoring the binding mode involving the slanted orientation, which increases vesicle fusogenicity. Interestingly, we observed a considerably stronger depression during a 10 Hz stimulus train for the R769E mutant than for the R603E mutant (Fig. 4). This finding supports the notion that accumulation of Ca 2+ during repetitive stimulation facilitates the transition to slanted orientations, which are expected to be destabilized by the R769E mutation but not by the K603E mutation.
It was surprising that the K720E mutation in the polybasic interface did not impair SV priming and enhanced Ca 2+ -evoked release as well as the Pvr, in contrast to the effects of the K603E in a nearby basic residue ( Fig. 2A-C). However, as mentioned above, there is likely a delicate balance between inhibitory and stimulatory interactions within the release machinery, and some of the interactions involving the polybasic region that are important for priming initiated by a perpendicular orientation of Munc13-1 need to be released to transit to slanted orientations that support full zippering of the SNARE complex and membrane fusion. Since the K720E mutation has only a modest effect on Ca 2+ -independent liposome binding and clustering (Fig. 7, 8D), a likely explanation of our results is that the mutant retains sufficient membrane affinity to fully support priming and facilitates the transition to the more active slanted orientations, yielding a higher release probability. This model is consistent with the finding that, in contrast to the K603E mutant, the K720E mutant exhibited less potentiation of release by PDBu than WT (Fig. 6A), likely because this mutation already promotes the slanted orientations that are favored by PDBu binding to the C1 domain. In 10 Hz stimulus trains, the K720E mutant depressed initially more than WT Munc13-1, but later exhibited analogous EPSC amplitudes as WT (Fig. 4D-F), likely because accumulation of Ca 2+ during repetitive stimulation already induces slanted orientations that do not involve interactions of K720 with the membrane. Nevertheless, we cannot rule out the possibility that the effects of the K720E 26 mutation arise from a different mechanism, for instance if the mutation alters an as yet unidentified interaction of Munc13-1.
The K706E mutation did not affect SV priming, in agreement with the notion that K706 does not participate in the interactions of the polybasic interface that mediate priming, but this mutant did decrease Ca 2+ -evoked release and hence the probability of release ( Fig. 2A-C). These effects are opposite to those caused by mutation of the corresponding lysine residue of Munc13-2 to Trp (the KW mutant) (Shin et al., 2010), which caused a gain-of-function, and could arise in principle because the K706E mutation hinders the transition to slanted orientations. This interpretation is consistent with the observation that EPSCs remained lower than WT for the K706E mutant during 10 Hz repetitive stimulation ( Fig. 4G-I). However, the observation that PDBu potentiation was similar for this mutant and WT Munc13-1 (Fig. 6A) suggests that the effects of the K706E mutation might not be related to the transition to slanted orientations but rather to another mechanism that directly influences fusion. For instance, the Munc13-1 C2B domain might cause membrane perturbations analogous to those that are believed to underlie the function of the Syt1 C2 domains in triggering release (Fernandez-Chacon et al., 2001;Rhee et al., 2005). It is also possible that the phenotypes caused by the K706E mutation and other mutations studied here reflect effects of Munc13-1 in more than one step leading to release, which complicates the interpretation of the data.
Despite these uncertainties in the interpretation of our results, the overall data strongly support the notions that the Munc13-1 C1C2B region plays a critical role in SV priming and that this region underlies two types of interactions with the plasma membrane that yield different orientations of Munc13-1. This model is also consistent with the previous observation that a mutation that is expected to unfold the Munc13-1 C1 domain leads to decreased priming but increased release probability in mice (Basu et al., 2007;Rhee et al., 2002), as the absence of the C1 domain is expected to impair the initial binding to the plasma membrane that is important for priming but likely helps to adopt slanted orientations that 27 facilitate release. Similarly, the finding that deletion of the C1 domain or the C2B domain of C. elegans unc-13 enhances release but deletion of both domains strongly impairs release (Michelassi et al., 2017) suggest that both the C1 domain and the C2B domain are important to stabilize the perpendicular orientations that mediate priming but hinder transition to the active, slanted orientations; however, at least one of the two domains needs to be present to mediate the interaction with the plasma membrane.
Clearly, further research will be necessary to better understand the nature of the LS and TS states involving different orientations of Munc13-1 and the factors that influence the equilibrium between them.
For instance, the equilibrium is most likely shifted toward the TS state by Syt1, which promotes trans-SNARE complex assembly, and by complexins, which stabilize assembled trans-SNARE complexes . However, binding of Syt1 and complexins to the SNARE complex is also believed to prevent final zippering of the very C-terminus of the SNARE complex before Ca 2+ influx, thus ensuring synchronicity of release upon Ca 2+ influx (Voleti et al., 2020). Other proteins such as CAPS, which has functions related to those of Munc13s (Jockusch et al., 2007), may also affect the equilibrium between the loose and tight primed states. Much we have learned but much remains to be learned about this fascinating system. (4.1%) SAPE, 21 (3.1%) SAGL. The crystal structure of the Munc13-1 C1C2BMUN fragment lacking residues 1408-1452 (which correspond to a long variable loop) (PDB code 5UE8) lacks multiple loops for which no density could be observed . To build a complete model of this fragment (except for residues 1408-1452), the coordinates of this crystal structure were merged with those of the missing loops in the NMR structure of the C1 domain (PDB code 1Y8F) (Shen et al., 2005), the crystal structure of the Ca 2+ -bound C2B domain (PDB code 6NYT) (Shin et al., 2010) and the refined crystal structure of the MUN domain (PDB code 5UF7) , after superimposing the common coordinates of these structures. Two additional missing loops were modeled with Robetta (Song et al., 2013) (https://robetta.bakerlab.org/). We placed the Ca 2+ -free C1C2BMUN model above the upper leaflet in an approximately perpendicular orientation with the polybasic face close to the membrane for the Ca 2+ -free simulation (black wire diagram in Figure 1-Figure supplement 1A). For the Ca 2+ -bound simulation, we built another system that included two Ca 2+ ions bound to the corresponding binding sites of the C2B domain (Shin et al., 2010) and where C1C2BMUN was placed in a more slanted orientation such that the membrane was close to the DAG-and Ca 2+ /PIP2-binding sites of the C1 and C2B domains, respectively (black wire diagram in Figure 1-Figure supplement 1C).

Molecular dynamics simulations.
All computations with these systems were performed with Gromacs 5.0.6 at the BioHPC supercomputing facility of UT Southwestern or with Gromacs 2019.6 at the Texas Advanced Computing Center using the CHARMM36 force field (Best et al., 2012). TIP3P explicit water boxes were built for both systems (24.3 x 26.7 x 24.3 nm 3 for the Ca 2+ -free system; 24.3 x 28.3 x 24.3 nm 3 for the Ca 2+ -bound system), and potassium and chloride ions were added to yield a KL concentration of 145 mM and overall charge neutrality, resulting in systems of 1.55 (Ca 2+ free) and 1.64 (Ca 2+ -bound) million atoms. The systems were energy minimized, heated to 310 K over the course of 1 ns in the NVT ensemble and equilibrated for 1 ns in the NPT ensemble. NPT production simulations were ran for 100 ns (Ca 2+ -free system) or 86 ns (Ca 2+bound system) using 2 fs steps and a 1.1 nm cutoff for non-bonding interactions, and periodic boundary conditions were imposed with Particle Mesh Ewald (PME) (Darden et al., 1993) summation for long-range electrostatics. Pymol (Schrödinger, LLC) was used for molecular graphics.

Hippocampal neuronal cultures
Single Munc13-1/2 DKO hippocampal neurons seeded onto micro-islands of astrocytes (autaptic hippocampal neurons) were prepared as previously reported (Bekkers and Stevens, 1991). Briefly, HClcleaned 30 mm diameter glass coverslips were coated with agarose type II-A (Sigma-Aldrich) and dried for more that 48 hrs. Pre-coated coverslips were printed by stamping (0.2 mm spot diameter and 0.5 mm spot interspace) with a cell-attachment mixture of rat tail collagen (BD Biosciences), poly-D-lysine (Sigma-Aldrich) and acetic acid (XX). Astrocytes were prepared from cerebral cortices of C57BL/6N mice of either sex at postnatal day 0-1 (P0-1). Cortices were isolated and digested with 0.25% trypsin-EDTA solution (Gibco) for 15 min at 37 °C. Enzymatic solution was discarded, and cortices were washed twice with Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) 30 (PAA) and 50 IU/ml penicillin and 50 µg/ml streptomycin (Gibco). After washing, tissue was homogenized in the same medium and cell suspension was cultured in 75 cm 2 flasks containing DMEM supplemented with FBS and penicillin/streptomycin. After 15 days in vitro (DIV) non astrocytic cells were removed, and astrocytes were trypsinized and plated onto the microdot printed coverslips at a density of 5000 cell/cm 2 .

Munc13-1 lentiviral rescue constructs
Munc13-1 control and point mutants were generated with a FLAG-tagged at the C-terminus. Munc13-1 cDNA was constructed from rat Unc13a splice variant by PCR amplification . The reverse primer harbors a 3xFLAG sequence (Sigma-Aldrich) to allow the expression analysis. The antifade medium (Polysciences Europe). Fixed neurons were imaged on a confocal laser-scanning 33 microscope Leica TCS SP8 with identical settings used for all samples. Neuronal cultures were visualized using a 63 × oil immersion objective. Images were acquired using Leica Application Suite X (LAsX) software at 1,024 × 1,024 pixels resolution using a z-series projection of 10-12 images with 0.3 μm depth intervals.
Six independent neurons per group for each cultured and two different cultures were imaged and analyzed using ImageJ software.

Electrophysiological Data Analysis and Statistics
Electrophysiological data were analyzed offline using Axograph X software version 1.4.3 (AxoGraph Scientific). Statistical analyses were carried out with GraphPad Prism software version 8 (GraphPad software). Number of neurons and cultures used for the statistical analyses are specified within the bar plots. Data was tested for normality by a D'Agostino-Pearson test showing a non-normal distribution.
Electrophysiological data are presented as normalized means to the corresponding WT control group ± standard error for means (SEM), except for the Pvr and PPR that are reported as absolute means ± SEM.
Statistical comparison between the different mutant groups was performed with Mann-Whitney U test.
The significance level was set at p = 0.05.
Plasmids and recombinant proteins. Plasmids used to express the following proteins, as well as methods for expression and purification in bacteria were described previously: full-length Homo sapiens SNAP-25A with and without its four cysteines mutated to serine, full-length Rattus norvegicus synaptobrevin-2, full- in E. coli were described previously (Chen et al., 2006;Dulubova et al., 2007;Dulubova et al., 1999;Liu et al., 2016;Ma et al., 2013;Prinslow et al., 2019;Sitarska et al., 2017;Stepien and Rizo, 2021  Liposome fusion assays. Liposome lipid and content mixing assays were performed as previously described (Liu et al., 2016;Liu et al., 2017;Stepien and Rizo, 2021). top. The proteoliposomes were spun at 4°C for 1.5 hours at 55,000 RPM in an SW-60 TI rotor and the top layer was collected. Concentrations of the final T-proteoliposomes were measured by the Stewart method (Stewart, 1980). V-proteoliposome concentrations were estimated from the UV-vis absorption using a standard curve made using known quantities of liposomes containing 1.5% NBD-PE. For the experiments of Figure 10- Figure supplement 1, we used the same protocol except for the following modifications. We used WT SNAP-25 that was dodecylated as described (Stepien and Rizo, 2021) and incorporated into the T-liposomes with P:L ratio 1:800 instead of SNAP-25 with its four cysteines mutated to serine, and syntaxin-1 was incorporated into the T-liposomes with P:L ratio 1:5,000. Instead of V-liposomes, we used VSyt1-liposomes containing 40% POPC, 6.8% DOPS, 30.2% POPE, 20% Cholesterol, 1.5% NBD PE, and 1.5% Marina Blue DHPE, synaptobrevin (1:10,000 P:L ratio) and synaptotagmin-1(57-421) C74S/C75A/C77S/C79I/C82L/C277S (P:L ratio 1,1000), as described (Stepien and Rizo, 2021).
To perform the fusion assays, T-liposomes (250 μM total lipid) were first incubated with 1 μM Munc18-1 (WT or D326K as indicated), 0.8 μM NSF, 2 μM αSNAP, 2 mM ATP, 2.5 mM Mg 2+ , 5 μM streptavidin, and 100 μM EGTA for 15-25 minutes at 37°C, and then were mixed with V-liposomes or To perform the fusion assays, T-liposomes (250 μM total lipid) were first incubated with 1 μM Munc18-1 wild type, 0.8 μM NSF, 2 μM αSNAP, 2 mM ATP, 2.5 mM Mg 2+ , 5 μM streptavidin, 100 uM EGTA, and 0.5 μM Fluo-4, pentapotassium salt for 15-25 minutes at 37°C. To initiate the reaction preincubated T-liposomes were mixed with V-liposomes (125 μM total lipid), 1 μM SNAP-25, and 0.1 μM C1C2BMUNC2C. After 5 minutes Ca 2+ was added to stimulate fusion, and 1% β-OG was added after 25 minutes to solubilize the liposomes. Throughout the reaction lipid mixing was monitored using DiD dequenching with excitation at 560 nm and emission measured at 670 nm. After solubilization of samples, the Fluo-4 emission intensity was measured from 505 to 540 nm with excitation at 465 nm. To measure the maximum fluorescence of Fluo-4 in each sample, 10 mM Ca 2+ was added to each sample and the fluorescence spectrum was acquired again. Ca 2+ concentrations were calculated as previously described (Grynkiewicz et al., 1985).  whereas the slanted orientation facilitates full assembly. The two states are proposed to exist in an equilibrium that is shifted to the right by Ca 2+ and DAG.              Fig. 7) were converted to average radii by calculating a populating weighted average, i.e. the sum of terms obtained by multiplying the radius of each bin by the corresponding population. Note that the equations used to derive the populations of each particle size bin break down for large radii. Therefore, these data need to be interpreted with caution, and the difference between the average radii calculated for the K720E C1C2BMUNC2C mutant in the absence and presence of Ca 2+ cannot be considered meaningful.         Munc13-1/2 DKO + Munc13-1 W T K 6 0 3 E K 7 2 0 E K 7 0 6 E R 7 6 9 E K 7 0 6 E / R 7 6 9 E K 6 0 3 E / K 7 2 0 E K 6 0 3 E / K 7 2 0 E K 7 0 6 E / R 7 6 9 E