Double mutation of open syntaxin and UNC-18 P334A leads to excitatory-inhibitory imbalance and impairs multiple aspects of C. elegans behavior

SNARE and Sec/Munc18 proteins are essential in synaptic vesicle exocytosis. Open form t-SNARE syntaxin and UNC-18 P334A are well-studied exocytosis-enhancing mutants. Here we investigate the interrelationship between the two mutations by generating double mutants in various genetic backgrounds in C. elegans. While each single mutation rescued the motility of CAPS/unc-31 and synaptotagmin/snt-1 mutants significantly, double mutations unexpectedly worsened motility or lost their rescuing effects. Electrophysiological analyses revealed that simultaneous mutations of open syntaxin and gain-of-function P334A UNC-18 induces a strong imbalance of excitatory over inhibitory transmission. In liposome fusion assays performed with mammalian proteins, the enhancement of fusion caused by the two mutations individually was abolished when the two mutations were introduced simultaneously, consistent with what we observed in C. elegans. We conclude that open syntaxin and P334A UNC-18 do not have additive beneficial effects, and this extends to C. elegans’ characteristics such as motility, growth, offspring bared, body size, and exocytosis, as well as liposome fusion in vitro. Our results also reveal unexpected differences between the regulation of exocytosis in excitatory versus inhibitory synapses.


Introduction
Sinusoidal C. elegans movement requires coordination between the excitation and inhibition of the neuromuscular junction (Jospin et al., 2009).Excitation/inhibition is initiated by the release of excitatory (acetylcholine) or inhibitory (gamma-aminobutyric acid, GABA) transmitters at their respective synapses via synaptic vesicle exocytosis (Richmond and Jorgensen, 1999).
Synaptic vesicle exocytosis consists of spontaneous and evoked exocytosis.While the fundamental mechanisms of exocytosis are well studied, the mechanisms that are specific to the types of synapses (excitatory vs. inhibitory) or types of exocytosis (spontaneous vs. evoked) are largely unknown.One notable exception is the complexin/CPX-1 protein, which in C. elegans blocks spontaneous exocytosis while enhancing evoked exocytosis (Hobson et al., 2011;Martin et al., 2011).SNARE (soluble NSF-attachment receptor) and Sec/Munc18 (SM) proteins are the essential proteins that execute the membrane fusion processes that underlies synaptic vesicle exocytosis.These proteins are conserved and play critical roles regardless of the types of synapses or types of exocytosis.The neuronal SNARE complex is comprised of the proteins syntaxin, synaptobrevin/VAMP and SNAP-25, coming together to form a four-helix bundle called the SNARE complex (Sutton et al., 1998;Rizo, 2022).In this complex, syntaxin and synaptobrevin each contribute one helix to the four-helix bundle, while SNAP-25 contributes two helices (Sutton et al., 1998).The C. elegans UNC-64 is known as syntaxin in mammalian systems, while the C. elegans UNC-18 is known as mammalian .The SNARE complex is tightly regulated by SNARE regulatory proteins, such as Munc18/UNC-18 (Hata et al., 1993;Verhage et al., 2000;Weimer et al., 2003), Munc13/UNC-13 (Nils et al., 1995;Augustin et al., 1999;Richmond et al., 1999) and the Ca 2+ sensor synaptotagmin-1/SNT-1 (Geppert et al., 1994;Jorgensen et al., 1995;Li et al., 2021).Together, these proteins work in harmony to fuse vesicles to the plasma membrane and release neurotransmitters in a Ca 2+dependent manner (Geppert et al., 1994;Fernández-Chacón et al., 2001).
Loss-of-function mutations of these essential exocytosis proteins lead to death or severe movement defects in C. elegans, mice, and humans.On the other hand, gain-of-function mutations lead to enhanced exocytosis and can rescue the phenotype of other exocytosis defective mutants.The investigation of such gain-of-function mutants provides mechanistic insights into how these proteins and their interactions with other exocytotic proteins contribute to exocytosis.Thus, an L166A/E167A mutation in syntaxin/UNC-64 (abbreviated as "LE") and a P335A mutation in Munc18/P334A mutation in UNC-18 (abbreviated as "PA") are well-known gain-of-function mutations that have been extensively studied in various model systems of exocytosis, including in vitro liposome fusion (Parisotto et al., 2014;Sitarska et al., 2017), PC12 cells (Han et al., 2014), C. elegans (Richmond et al., 2001;Park et al., 2017;Tien et al., 2020) and mice (Gerber et al., 2008;Munch et al., 2016).Both mutants exhibited enhanced fusion/exocytosis in every assay system tested.However, the interaction between the two mutations has not been investigated.
The t-SNARE syntaxin/UNC-64 exists in two conformations, the open and the closed conformations (Dulubova et al., 1999).The LE mutation in syntaxin leaves syntaxin constitutively open, thus facilitating SNARE complex formation (Dulubova et al., 1999;Gerber et al., 2008) and was originally considered to selectively rescue unc-13 and unc-10/RIM mutants in C. elegans, without enhancing exocytosis on its own (Richmond et al., 2001).This led to the hypothesis that the role of Munc13/UNC-13 is to open syntaxin at the synapse (Koushika et al., 2001;Richmond et al., 2001), which was later support by biophysical assays in vitro (Ma et al., 2011).However, later work found that open syntaxin 1B knock-in (KI) mice exhibit enhanced spontaneous and evoked release from cortical neurons (Gerber et al., 2008).Similarly, our recent generation of open syntaxin knock-in worms revealed that this gain-of-function mutation 4 enhances excitatory synaptic transmission on its own and it can rescue a variety of exocytosisdefective mutants, including synaptotagmin-1/snt-1, unc-2 and CAPS/unc-31, as well as unc-13 and unc-10 in C. elegans (Tien et al., 2020).These findings showed that the facilitation of SNARE complex assembly caused by the LE mutation enhances neurotransmitter release in a variety of genetic backgrounds (Dulubova et al., 1999;Ma et al., 2011).
Munc18/UNC-18 has an arched structure comprised of three domains that form a central cavity.
This central cavity is where syntaxin binds when syntaxin is in a closed conformation (Misura et al., 2000).Formation of this binary complex mediates syntaxin trafficking/chaperoning to the plasma membrane (Arunachalam et al., 2008;Han et al., 2009;Han et al., 2011;Han et al., 2014).In addition to syntaxin, Munc18/UNC-18 can bind to synaptobrevin, forming a template for SNARE complex assembly (Parisotto et al., 2014;Baker et al., 2015).The P334A mutation in Munc18/UNC-18 was designed to extend a helix that binds to synaptobrevin and thus enhance binding, but such enhancement was not observed (Parisotto et al., 2014), and synaptobrevin binding does not require extension of this helix (Stepien et al., 2022).Instead, the P334A mutation impairs syntaxin binding (Han et al. 2014) and causes a gain-of-function because the release of contacts between syntaxin and Munc18/UNC-18 at this site is important to form the template complex and initiate SNARE complex assembly (Stepien et al. 2022).Thus, similar to the open syntaxin, P334A knock-in worms exhibit enhanced excitatory synaptic transmission on its own, and rescues unc-31 and unc-13 mutant worms (Park et al., 2017).
In the present study, we investigated how the open syntaxin and P334A UNC-18 mutations interact with each other using C. elegans as a model system.We initially anticipated that the double mutant would exhibit additive or synergistic effects on exocytosis, as well as enhanced ability to rescue motility and other altered characteristics observed in various C. elegans exocytosis mutants.To our surprise, the double mutants exhibit suppressed motility with enhanced acetylcholine release, as measured by thrashing assays and the sensitivity to the acetylcholinesterase inhibitor (aldicarb), respectively.Strikingly, the presence of both gain-offunction mutations within the same worm did not provide benefits to worm size, growth speed, or number of offspring given.Therefore, we further analyzed these single and double mutants by electrophysiology in detail.Our results show that these mutants differentially contribute to exocytosis depending on the type of synapse and the type of exocytosis.31, unc-13 and unc-10 (Tien et al., 2020).Although we showed that the unc-18(PA) KI mutant rescues unc-31 and unc-13 mutants (Park et al., 2017), whether unc-18(PA) has the ability to rescue a wide-range of exocytosis mutants remains unknown.
Snt-1(md290) worms have smaller brood sizes, body sizes and exhibit slow population growth speeds (Jorgensen et al., 1995;Li et al., 2021).Therefore, we also tested whether unc-64(LE), unc-18(PA), or tom-1(ok285) mutations can rescue the reduced brood size, population growth speed, and body sizes observed in snt-1 null worms.For brood size, a single L4 worm was placed on an agar plate and after 48 hours of growth, the original worm was transferred to a fresh agar plate.Afterwards, the original worm was transferred to a fresh new plate every 24 hours until the worm died, or egg laying ceased.Offspring (eggs laid and already hatched worms) were counted after the original worm was removed from the plate.We found that unc-64(LE) and unc-18(PA) worms both had comparable brood sizes to that of N2, while tom-1 had a significantly reduced brood size (Fig. 1D).snt-1 null worm brood sizes were even smaller than the brood size of tom-1 worms (Fig. 1D), and when we crossed the various mutants into the snt-1 null background, although there was a trend for rescue by the snt-1; unc-18(PA) double mutant, the double mutation of snt-1; tom-1 seemed to worsen the small brood size of snt-1 null worms (Fig. 1D).
For the population growth assay, three worms of each strain were placed on agar plates and allowed to grow over the course of 9 days, and L4 and above adult worms were counted daily.N2 worms showed a sharp increase in total worm number between days 3 and 4, reaching a peak at day 5 (Fig. 1F).Snt-1 null worms lagged and did not peak in population until day 8 showing a slow rate of population growth.Both snt-1; unc-64 (LE) and snt-1; unc-18(PA) worm populations grew faster than snt-1 null worms.The rescue observed by open syntaxin was stronger than that of the P334A UNC-18 mutant; snt-1; unc-64(LE) worms reached their peak a day earlier than snt-1; unc-18(PA) worms.However, snt-1; tom-1 worms displayed decreased growth speeds compared to the snt-1 null mutant.
Despite finding no significant rescue in brood size by either snt-1; unc-64(LE) or snt-1; unc-18(PA), we did observe an increase in populational growth of these two strains when compared to snt-1 null worms (Fig 1F).This discrepancy could be explained by snt-1; unc-64(LE) worms giving birth to a larger portion of their offspring earlier on.Indeed, when we plotted the brood counts by day (Fig. 1E), we observed that while snt-1; unc-64(LE) worms and snt-1;unc-18(PA) worms had a similar cumulative brood counts, snt-1; unc-64(LE) worms had a left-shifted curve and laid more eggs in the beginning.These worms that were laid earlier (F1 progeny) would then mature and give rise to the F2 progeny that may be reflected in the rapid peak observed in the population growth assay (Fig. 1F).
Next, we analyzed worm body size.For this, C. elegans were synchronized and imaged two days after they reached the L4 stage.The captured image of the worm was manually traced to find the perimeter of the worm.We found that snt-1 null worms showed a smaller body size compared to N2 worms (Fig. 1G).While unc-64(LE) weakly increased the size of snt-1 null worms, unc-18(PA) did not increase the size of snt-1 null worms.We found that tom-1 null single mutants also exhibited a smaller body size, which is consistent with previous literature (Lee et al., 2011), and snt-1;tom-1 worms resulted in a further significant decrease to the size of the worm.These results may suggest that hypersecreting mutants such as tom-1 (Lee et al., 2011), open syntaxin, and P334A UNC-18 (Park et al., 2017) may have a common phenotype of having a smaller body size.Overall, we found that open syntaxin and P334A UNC-18 have similar rescuing abilities on motility, acetylcholine release, brood size and growth speed on snt-1 null mutants.Such rescuing effects are absent in the tom-1 null mutant.The results also suggest that open syntaxin and P334A UNC-18 utilize similar mechanisms to enhance exocytosis, which is distinct from the exocytosis-enhancing mechanism exhibited by the lack of the inhibitory tomosyn/TOM-1 protein.

Simultaneous P334A UNC-18 and open syntaxin abolish the ability to rescue motility and growth speed of snt-1 mutants but rescues aldicarb sensitivity
We found that open syntaxin and P334A UNC-18 mutants can individually rescue the motility, brood size, and growth speed of snt-1 mutants, and that this rescue is accompanied by an increase in acetylcholine release.What remains unknown is if/how the rescuing effects of the two mutants are interrelated.If open syntaxin and P334A UNC-18 use independent mechanisms to enhance exocytosis of snt-1 null, we would anticipate that the unc-64(LE) and unc-18(PA) double mutation may have additive effects when rescuing the snt-1 null mutant.On the other hand, if they use common or similar mechanisms, we may see saturating effects between the two.Therefore, we next asked if the double mutation would exhibit an additional ability to rescue motility and acetylcholine release in the snt-1 null background.As such, we generated the snt-1; unc-64(LE); unc-18(PA) triple mutant.Despite both open syntaxin and P334A UNC-18 mutants being able to rescue thrashing activity individually (Fig. 1A), the snt-1; unc-64(LE); unc-18(PA) triple mutant worms showed a similar thrashing count to the snt-1 null mutant (Fig. 2A).The increased brood size and population growth rate seen by snt-1; unc-64(LE) and snt-1; unc-18(PA) double mutants were also abolished in the triple mutant -snt-1; unc-64(LE); unc-18(PA) triple mutant worms showed a trend for reduced brood size (Fig. 2C), and a similar growth rate to that of the snt-1 null worm (Fig. 2D).Importantly, however, the triple mutant was still able to show a strong rescue of aldicarb sensitivity (Fig. 2B).Overall, we surprisingly find that the behavioral benefits gained by the single open syntaxin or single P334A UNC-18 mutants are lost when both mutations are present simultaneously; yet they strongly rescued aldicarb sensitivity.
Thus, the triple mutant exhibits a striking dissociation between behavior and aldicarb sensitivity.

The P334A UNC-18 and open syntaxin double mutant dramatically worsens the motility of unc-31 while rescuing aldicarb sensitivity
We next tested the two knock-in mutations in a different background to see if the loss of beneficial effects of the combined mutations is specific to the snt-1 null background.
Since we found detrimental effects in motility when simultaneously expressing unc-64(LE) and unc-18(PA) mutations in snt-1 null and unc-31 null backgrounds, we hypothesized that the double mutation may induce motility defects in a wild-type background.Thus, we crossed unc-64(LE) worms with unc-18(PA) worms to look at the resulting phenotype of the double mutants in a wild-type background.We observed that both single unc-64(LE) and unc-18 (PA) mutants thrashed at a degree comparable to N2.However, the unc-64(LE); unc-18(PA) double mutants exhibited a dramatically decreased thrashing, with only 65 thrashes/min, which is about half of the wild-type thrashing (Fig. 3D).As previously shown, we found that unc-64 (LE) or unc-18(PA) single mutants display increased aldicarb sensitivity compared to N2 (Park et al., 2017;Tien et al., 2020).At the usual 1 mM concentration of aldicarb, there was no further increase observed by the double mutant of unc-64 (LE); unc-18(PA) compared to the unc-18(PA) single mutant (Fig. 3E).However, at a lower (0.3 mM) concentration of aldicarb, we observed an increase in aldicarb sensitivity in the double mutant compared to the two single mutants (Fig. 3F).From these experiments (Fig. 1, 2, 3), we conclude that independently, unc-64(LE) and unc-18(PA) have beneficial effects on the motility, exocytosis, brood, and growth speed of exocytosis-defective mutants, such as snt-1 or unc-31 null mutants.However, when these mutants are present simultaneously, their beneficial effects are lost or become detrimental regardless of the presence or absence of exocytosis-defective mutations.Importantly however, the double mutant increased aldicarb sensitivity irrespective of the background.

P334A UNC-18 enhances excitatory synaptic transmission like open syntaxin but the enhancement is lost in the double mutant
Why do the double mutants exhibit reduced motility while it increases or rescues aldicarb sensitivity (Fig. 1 -3)?We envisioned two scenarios for these findings: in the first scenario, we hypothesized that the double mutation causes a severe imbalance of excitation over inhibition; that is, the double mutation selectively enhances acetylcholine release at the excitatory synapse while decreasing GABA release at the inhibitory synapse.In the second scenario, we hypothesized that the double mutation selectively increases spontaneous release while decreasing evoked transmitter release.Such phenomenon was previously shown in cpx-1 null mutants, which exhibit reduced motility but enhanced aldicarb sensitivity (Hobson et al., 2011;Martin et al., 2011).Therefore, to reveal the underlying mechanism of the paradoxical phenotype of the double mutant, we decided to examine spontaneous and evoked neurotransmitter release from both excitatory and inhibitory synapses from wild-type control C. elegans, the single mutants unc-64(LE) and unc-18(PA), and the double unc-64(LE); unc-18(PA) mutant.To measure evoked release from either excitatory or inhibitory synapses, we generated the respective mutant lines in the zxIs6 or zxIs3 background (Liewald et al., 2008;Tien et al., 2020).zxIs6 and zxIs3 strains express channelrhodopsin-2 in excitatory cholinergic neurons (zxIs6) or inhibitory GABAergic neurons (zxIs3), enabling optogenetic stimulation for accessing transmitter release.
First, recordings were performed in the absence of light stimulation to identify excitatory spontaneous release using the single and double mutant strains generated in a zxIs6 background.
Both unc-64(LE) and unc-18(PA) single mutants increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) (Fig. 4A, C), congruent with what we found previously (Park et al., 2017;Tien et al., 2020).While the unc-64(LE); unc-18(PA) double mutant exhibited a trend for increase in mEPSC frequency (Fig. 4A, B, table 1), the averaged value was lower than both of the single mutants and did not reach statistical significance (p = 0.138).This indicates that the unc-64(LE) and unc-18(PA) mutations do not have additive effects on mEPSC frequency.The amplitude of the spontaneous mEPSCs was not significantly changed across all strains (Fig. 4B).Next, we applied optogenetic stimulation (10 ms, blue light) and measured excitatory evoked postsynaptic currents (EPSCs).Similar to previous reports (Tien et al., 2020), unc-64(LE) mutants showed an increase in charge transfer of excitatory postsynaptic currents (EPSCs) without affecting the EPSC amplitude when compared to wild-type worms (Fig. 4D-H).unc-18(PA) single mutants exhibited a similar EPSC phenotype to unc-64(LE) and increased charge transfer without largely affecting EPSC amplitude (Fig. 4D-H).However, when unc-64(LE) and unc-18(PA) were present together, the two gain-of-function mutations again seemed to cancel each other out.The unc-64(LE); unc-18(PA) double mutant only showed a trend for increase in EPSC charge transfer and again values were much lower than each of the single mutants and did not reach statistical significance (Fig. 4D-H).While no significant changes to evoked EPSC amplitude was observed, we note that in the unc-64(LE); unc-18(PA) double mutant, the double mutant was the only strain to exhibit a decrease in amplitude values (Table 1).Thus, we conclude that the double mutant exhibits both a weak increase in spontaneous and evoked release than the single mutants at the excitatory synapse.

P334A UNC-18 enhances inhibitory synaptic transmission while open syntaxin and the double mutant do not
We then investigated inhibitory synaptic exocytosis using the single and double mutant strains in a zxIs3 background.We found that, in the absence of light stimulation, the frequency of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) was slightly increased in unc-64(LE) and unc-18(PA) worms (Fig. 5A, 5C).However, unlike mEPSCs, the frequency of mIPSCs of the unc-64(LE); unc-18(PA) double mutant did not show an increase (Fig. 5A, 5C, table 1).Again, amplitudes of mIPSCs were unchanged across all strains (Fig. 5B).We then looked at evoked inhibitory postsynaptic currents (IPSCs) by optogenetically stimulating the GABAergic neurons.We found that only the P334A UNC-18 mutant, unc-18(PA), facilitated IPSC charge transfer and amplitude, but this facilitation was lost in the unc-64(LE); unc-18(PA) double mutant (Fig. 5D-H).Not only was the facilitation lost, there was actually a decrease in averaged changes to both normalized and unnormalized amplitude and charge transfer (table 1).
Thus, the double mutant fails to enhance spontaneous and evoked transmitter release in GABAergic inhibitory synapses, and may further emphasize inhibitory effects.Together with the data of excitatory transmitter release (Fig. 4), we find that the double mutant of open syntaxin and P334A UNC-18 causes an imbalance between excitatory and inhibitory transmission.This may explain the phenotype we observed where double mutants exhibited higher aldicarb sensitivity with reduced motility.

Discussion
We found that the double mutation of open syntaxin and P334A UNC-18 has complex effects on exocytosis depending on the type of (excitatory vs. inhibitory) synapse and on the type (spontaneous vs. evoked) of exocytosis (Table 1).We first showed that P334A UNC-18 rescues detrimental phenotypes of the null snt-1(md290) mutant, a major Ca 2+ sensor protein for exocytosis (Fig. 1), similar to what we observed in open syntaxin mutants (Tien et al., 2020).We also found that similar to open syntaxin, P334A UNC-18 can also enhance exocytosis in a wide range of genetic backgrounds, in addition to rescuing unc-13 and unc-31 null mutants (Park et al., 2017), P334A UNC-18 also rescues snt-1 and unc-31 mutants.Thus implying that P334A may provide a general means to enhance synaptic transmission in normal and disease states.
Importantly however, the removal of tomosyn/TOM-1 did not rescue the exocytosis-deficient phenotype of snt-1 null worms (Fig. 1).Tomosyn is considered to inhibit exocytosis by forming the inhibitory tomosyn-SNARE complex, and tom-1 null worms enhance exocytosis by suppressing the formation of this inhibitory SNARE.On the other hand, open syntaxin and P334A UNC-18 enhance exocytosis by facilitating active SNARE assembly.Therefore, our results suggest that facilitation of the active SNARE assembly, but not the removal of inhibitory SNARE assembly, is needed to bypass the lack of synaptotagmin.This may imply that synaptotagmin is involved in active SNARE assembly.

Motivated by the rescue of a wide range of exocytosis-defective mutants by open
syntaxin and P334A UNC-18 individually, we investigated the potential additional beneficial effects that could be caused by introducing both mutations simultaneously.However, we found that regardless of the genetic background, the double mutant had detrimental effects on many characteristics of the C. elegans we tested, despite them showing an increased sensitivity to the acetylcholinesterase inhibitor, aldicarb (Fig. 1 -3).Although the coincident occurrence of open syntaxin and P334A UNC-18 increased acetylcholine release, worms harboring both mutations did not receive benefits to motility, body size, brood size, or growth speed.
Historically, in forward genetic screens, a large number of exocytosis defective mutants were isolated due to their resistance to cholinesterase inhibitors, which include aldicarb (Brenner, 1974;Nguyen et al., 1995;Miller et al., 1996).Therefore, aldicarb resistance and movement defects, such as the uncoordinated phenotype, are usually correlated in exocytosis mutants.In this aspect, our finding of the dissociation between motility and aldicarb sensitivity of the double mutant was surprising.This dissociation is particularly evident in the unc-31 null background.
Here, the animals exhibit aldicarb sensitivity similar to wild-type N2 levels, yet their thrashing is severely impaired (Fig. 3).This led us to study both excitatory and inhibitory synaptic transmission of the respective mutants in detail using electrophysiology combined with optogenetics (Fig. 4-7).As far as we know, this kind of detailed electrophysiological investigation in both spontaneous and evoked release in C. elegans excitatory and inhibitory synapses using optogenetics is unprecedented.
We summarized the synaptic transmission phenotypes of open syntaxin, P334A UNC-18 and the double mutant in the wild-type background and in the unc-31 null background in Table 1.P334A UNC-18 consistently increased responses in excitatory and inhibitory evoked release in the wild-type and unc-31 null background.Open syntaxin facilitated excitatory spontaneous and evoked release in both backgrounds, however, increases to inhibitory spontaneous and evoked transmission were weaker.The double mutant mirrored P334A UNC-18 in excitatory transmission and enhanced excitatory evoked charge transfer in both backgrounds.Strikingly however, the double mutant reduced spontaneous and evoked inhibitory release in both backgrounds.The differential effects of the double mutation on excitatory versus inhibitory synaptic transmission seem to explain why the double mutation worsened the motility of unc-31 while rescuing aldicarb sensitivity.In other words, the imbalance of excitatory over inhibitory synaptic transmission increases acetylcholine release at the C. elegans neuromuscular junctions and increases aldicarb sensitivity, while impairing motility and other C. elegans features such as motility, brood size, or body size (Fig. 8).Our results also highlight the importance of investigating excitatory and inhibitory transmission when phenotypically examining exocytosis mutants in the future.
The proteins which play key roles in synaptic vesicle exocytosis have been identified.We believe the next key question is to understand how these proteins interact with each other to achieve the precise regulation of synaptic vesicle exocytosis.To answer this question, it is important to examine their genetic interactions by generating double, triple and quadruple mutants.In this aspect, C. elegans as a model organism has significant advantages over mammalian systems.In mammals, closely related isoforms (e.g., syntaxin-1A, 1B; Munc13-1, 2; Tomosyn-1, 2) are present for each protein, and the compensatory and/or redundant effects of these isoforms makes it very difficult to evaluate the function of each protein (Fujiwara et al., 2006;Kofuji et al., 2014;Mishima et al., 2014).Conversely, even single knockout of certain proteins (e.g., syntaxin-1B, Munc18-1, Munc13-1) can cause embryonic or perinatal death in mice, which makes it virtually impossible to generate and analyze double or triple knockouts in mice.Thus, we believe that the detailed analysis of various C. elegans mutants using behavioral approaches and electrophysiology will keep providing new insights regarding the mechanisms of synaptic transmission as well as their relationship to behavior and other attributes C. elegans life.
(E) Brood size of indicated worm strains, snt-1 worms (red) decrease in brood size when compared to N2 (black), snt-1; unc-64(LE) in blue and snt-1;unc-18(PA) in dark green have a trend for rescuing the brood size of the worm.
(F) Number of worms and eggs laid by each worm strain by day, both snt-1; unc-64(LE) in blue and snt-1;unc-18(PA) in dark green increase the worms and eggs laid while snt-1;tom-1 decreases the worms and eggs laid.
(F) EPSC amplitude when calibrated with membrane capacitance to account for differences in animal size also did not show significant changes (one-way ANOVA, F (3, 52) = 0.9039 p = 0.4456).
(H) Charge transfer of EPSCs calibrated to membrane capacitance to account for differences in animal size, charge transfer again is increased for both unc-64(LE) and unc-18(PA) single mutants(F (3, 52) = 11.98;p < 0.0001).An trend of increase is also seen in EPSC charge transfer in the unc-64(LE);unc-18(PA) double mutant but again did not reach statistical significance (p = 0.2172)    Motility of each strain was determined by counting the thrashing rate of C. elegans in liquid medium.Post L4 young adult worms were placed in 60 μL of M9 buffer on a 35 mm petri dish lid.Worms were recorded for 4 minutes using an OMAX A3580U camera on a dissecting microscope with the OMAX ToupView v3.7 software.Worms were allowed to recover for 2 minutes and the later 2 minutes of each recorded video was used for thrashing analysis.Thrashes per minute was manually counted and averaged within each strain.A minimum of 40 worms per strain were used for analysis.A thrash was defined as a complete bend in the opposite direction at the midpoint of the body.

Aldicarb assays
Aldicarb sensitivity was assessed using synchronously grown adult worms placed on non-seeded 35 mm NGM plates containing 0.3 mM or 1 mM aldicarb.All assays were done in 1 mM aldicarb plates unless specified otherwise.Over a 4 hour period, worms were monitored for paralysis at 15 or 30 min intervals, worms were checked a final time 24 hours later.Worms were considered paralyzed when there was no movement or pharyngeal pumping in response to three taps to the head and tail with a platinum wire.
Once paralyzed, worms were removed from the plate.Six to eight sets of approximately 15 worms were examined for each strain.

Worm size
Using synchronized worms, two days past the L4 stage, worms were placed in M9 liquid buffer with 50 mM sodium azide to prevent the worm from moving during image acquisition.The worm was imaged using a Nikon scope and then the perimeter of the worm was determined.The perimeter of the worm was manually traced using ImageJ.20 worms were averaged for each strain.

Population growth
From a synchronized population of worms, three young adult worms were moved to a new plate containing OP50.No worms with visibly present eggs were selected to ensure synchronicity.The number of L4 and above worms were counted daily until the whole plate reached starvation.Each strain was repeated a minimum of 6 times.All worms were grown in the same box on the same shelf through the duration of the experiment.
UNC-18, but not the absence of TOM-1, rescues motility, acetylcholine release, and growth speed of snt-1 null mutants like open syntaxin We previously showed that an open syntaxin unc-64(LE) KI mutant can rescue the defects in motility and acetylcholine release of various exocytosis mutants, including snt-1, unc-

Figure captions Figure 1 .
Figure captions

Figure 2 .
Figure 2. Double mutations of P334A UNC-18 and open syntaxin abolish the ability to rescue motility and growth speed of snt-1 while they rescue aldicarb sensitivity

Figure 4 .
Figure 4. Optogenetic stimulation of P334A UNC-18 and open syntaxin show the double mutant does not facilitate excitatory synaptic transmission like the single mutants

Figure 5 .
Figure 5. P334A unc-18 enhances inhibitory synaptic transmission while open syntaxin and the double mutant do not

Figure 8 .
Figure 8. Summary of findings in open syntaxin, P334A UNC-18, and the simultaneous double mutant.