We used gene targeting to create mice that lack syntaxin-1A (syntaxin-1A^KO), and contain the ‘LE’ mutation in syntaxin-1B which renders it predominantly open (syntaxin-1B^Open; Fig. S1 [⁹]). Studying littermate offspring from crosses of double-heterozygous syntaxin-1A^KO and -1B^Open mice, we found that homozygous syntaxin-1A^KO mice exhibited no decrease in survival (Fig. 1A) or other obvious phenotypes (Figs. S2 and S3). The expendability of syntaxin-1A was unexpected in view of its high concentrations and proposed central functions (e.g., ^10-14), indicating that syntaxin-1A may be functionally redundant with syntaxin-1B.

Homozygous mutant syntaxin-1B^Open mice were also viable, but severely ataxic, and developed lethal epileptic seizures after 2 weeks of age (Figs. 1A and S3). The seizure phenotype of syntaxin-1B^Open mutant mice was recessive and independent of the syntaxin-1A^KO. Thus, syntaxin-1B was selectively essential, probably because it is more widely expressed than syntaxin-1A (¹⁵). In C. elegans, transgenic syntaxin-1^Open rescues the paralysis of unc-13 mutant worms (¹⁶), but crossing syntaxin-1B^Open mice with Munc13-1 knockout mice did not rescue the Munc13-1 knockout-induced lethality (Fig. S4).

The syntaxin-1A^KO mutation abolished syntaxin-1A expression (Fig. 1B), whereas the syntaxin-1B^Open mutation decreased syntaxin-1B levels (Fig. 1C). Both mutations produced a modest decrease in Munc18-1 levels, but no major changes in other proteins (Table S1). The syntaxin-1^Open mutation decreases formation of the Munc18-1–syntaxin-1 complex, but not formation of SNARE-complexes or Munc18-1–SNARE-complex assemblies (^3,8; Fig. S5). Consistent with this conclusion, less Munc18-1 was co-immunoprecipitated with syntaxin-1 in syntaxin-1B^Open mice, whereas other SNARE-proteins co-immunoprecipitated normally (Figs. 1D and S6; note that SNARE-complex–Munc18-1 assemblies are not stable during immunoprecipitations, and thus cannot be evaluated).

Electron microscopy of cultured cortical neurons from littermate syntaxin-1B^Open and -1B^WT mice lacking syntaxin-1A revealed increased vesicle docking in syntaxin-1B^Open synapses (~25% increase; Figs. 2A-2D). The size of the postsynaptic density was also increased (~20%; Fig. 2E), while the density of docked vesicles per active zone length was unchanged (Fig. 2F). No other structural parameter measured differed between syntaxin-1B^Open and -1B^WT synapses; in particular, the number and intraterminal distribution of vesicles were unaltered (Fig. S7). In chromaffin cells, however, the syntaxin-1B^Open mutation caused a large decrease in chromaffin vesicle docking similar to the Munc18-1 knckout. Again, neither mutation altered the total number of chromaffin vesicles (Figs. 2K and 2L). Synaptobrevin-2 KO mice, analyzed in parallel as a negative control, did not change chromaffin vesicle docking, but increased the total number of chromaffin vesicles (Fig. 2L). Consistent with earlier findings (^17-20), these results indicate that the Munc18-1–syntaxin-1 complex, but not the SNARE complex, functions in chromaffin vesicle docking. This function may not be apparent in vertebrate synapses because active zone proteins that are absent from chromaffin cells probably dock synaptic vesicles independent of their attachment to the Munc18-1–syntaxin-1 complex.

Measurements of spontaneous miniature excitatory postsynaptic currents (mEPSCs), of excitatory postsynaptic currents (EPSCs) evoked by isolated action potentials, and of use-dependent synaptic depression during high-frequency stimulus trains in hippocampal neurons revealed no significant difference between syntaxin-1A^KO and wild-type synapses (¹²). In syntaxin-1B^Open synapses (on the syntaxin-1A^KO background), however, the mEPSC frequency was increased ~40%, and use-dependent depression of EPSCs was massively enhanced, although – surprisingly – evoked EPSCs exhibited a normal amplitude and kinetics (Fig. 3 and Fig. S8).

The increased depression in syntaxin-1B^Open synapses indicates that they exhibit a decreased readily-releasable vesicle pool (RRP), an increased release probability, and/or a decreased rate of refilling of the RRP after it is emptied. To test this, we measured the RRP by application of 0.5 M sucrose (^9,21). The RRP was unchanged in syntaxin-1A^KO synapses, but decreased ~35% in syntaxin-1B^Open synapses (Figs. 4A and 4B), consistent with the decreased syntaxin-1 and Munc18-1 levels in syntaxin-1B^Open mice (Fig. 1C). Determination of the RRP size in the same synapses in which we monitored mEPSCs and evoked EPSCs (Figs. 3A-3F) allowed us to calculate the spontaneous vesicular release rate (as the ratio of mEPSC frequency to the RRP) and the vesicular release probability P_vr (as the ratio of EPSC and RRP charges). Both were increased >2-fold in syntaxin-1B^Open synapses (Figs. 4C and 4D), augmenting the percentage of the RRP released by a single action potential from ~10% in syntaxin-1B^WT to ~20% in syntaxin-1B^Open synapses. Measurements of the refilling rate of the RRP, however, detected an increase, and not a decrease (Fig. S9).

Thus, opening syntaxin-1 facilitates the fusion of synaptic vesicles on the background of a smaller RRP without changing the recruitment of vesicles into the RRP. Consistent with this conclusion, we found that the syntaxin-1B^Open mutation accelerates sucrose-induced release (Figs. 4E and 4F), and significantly boosts the relative amount and fractional release rate induced at lower sucrose concentrations (Figs. 4G-4I). Moreover, the syntaxin-1B^Open mutation increases the apparent Ca²⁺-sensitivity of neurotransmitter release (Fig. S10), and occludes the phorbol-ester induced potentiation of release (Fig. S11). Overall, these results establish that although the RRP is smaller in syntaxin-1B^Open synapses, their resident RRP vesicles are more fusogenic.

Here, we show that the ‘closed’ conformation of syntaxin-1 performs three functions upstream of the canonical role of syntaxin-1 as a SNARE-protein in membrane fusion:

Ca²⁺ and sucrose trigger fusion of primed synaptic vesicles. Primed vesicles are thought to be suspended in a metastable state in which SNARE-complexes are assembled but the bilayers have not yet fused (²²). We propose that primed vesicles are associated with a variable number of assembled SNARE-complexes, and that this number dictates the sucrose- and Ca²⁺-sensitivity of a given vesicle (see model in Fig. S12). To account for the synaptic phenotype of syntaxin-1B^Open mutant mice, we hypothesize that the syntaxin-1B^Open mutation increases the average number of assembled SNARE-complexes per vesicle, and thereby enhances their Ca²⁺- and sucrose-sensitivity. On the other hand, the destabilization of syntaxin-1 and Munc18-1 by the syntaxin-1B^Open mutation (Fig. 1) decreases the total number of primed vesicles and thus the RRP, even though the primed vesicles are more fusogenic. The decrease in RRP is not due to the increased spontaneous release rate because its spontaneous fusion rate is still >100-fold lower than the vesicle repriming rate, and because much higher spontaneous fusion rates in synaptotagmin-mutant mice do not decrease the RRP size [²³]). An alternative hypothesis would be that primed vesicles lack assembled SNARE-complexes, and Ca²⁺ or hypertonic sucrose trigger fusion of primed vesicles by inducing the opening of syntaxin-1 and assembly of SNARE-complexes (Fig. S12). The simplicity of this second model is attractive, but it cannot account for the speed of Ca²⁺-triggered fusion, or for its dependence on complexin which binds to assembled SNARE complexes (²²). Independent of which model is correct, our results demonstrate that syntaxin-1 performs multiple functions in exocytosis that go beyond its role as a SNARE-protein to include the control of vesicle docking and the regulation of the vesicle fusion rate.

supplementary data