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During synaptic vesicle fusion, the SNARE-protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a ‘closed’ conformation outside the SNARE-complex, and an ‘open’ conformation in the SNARE-complex. Whereas SNARE-complexes containing ‘open’ syntaxin-1 and Munc18-1 are essential for exocytosis, the significance of ‘closed’ syntaxin-1 is unknown. Here, we generated knockin/knockout mice that expressed only ‘open’ syntaxin-1B. Syntaxin-1BOpen mice were viable, but succumbed to generalized seizures at 2-3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1BOpen synapses, and the size of the readily-releasable vesicle pool was decreased, whereas the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.
Intracellular membrane fusion reactions are carried out by interactions between SNARE- and SM-proteins (for ‘Soluble NSF-Attachment Protein Receptors’ and ‘Sec1-Munc18 like proteins’; 1,2). In Ca2+-triggered exocytosis in neurons and neuroendocrine cells, fusion is catalyzed by formation of SNARE complexes from syntaxin-1, SNAP-25, and synaptobrevin/vesicle-associated membrane protein, and the binding of the SM-protein Munc18-1 to these SNARE complexes (1-3). Syntaxin-1 consists of two similar isoforms (syntaxin-1A and -1B) that are composed of an N-terminal α-helical domain (the Habc-domain) and a C-terminal SNARE-motif and transmembrane region. Outside of the SNARE-complex, syntaxin-1 assumes a ‘closed’ conformation in which the Habc-domain folds back onto the C-terminal SNARE motif (4,5). In the SNARE-complex, by contrast, syntaxin-1 is ‘opened’ (6). Munc18-1 interacts with syntaxin-1 alone in the ‘closed’ conformation to form heterodimers (3,4), and additionally binds to SNARE-complexes containing syntaxin-1 in the ‘open‘ conformation to form SNARE-complex/Munc18-1 assemblies (7,8) which are essential for exocytosis (3). The significance of the closed conformation of syntaxin-1 and its binding to Munc18-1 remain unknown.
We used gene targeting to create mice that lack syntaxin-1A (syntaxin-1AKO), and contain the ‘LE’ mutation in syntaxin-1B which renders it predominantly open (syntaxin-1BOpen; Fig. S1 ). Studying littermate offspring from crosses of double-heterozygous syntaxin-1AKO and -1BOpen mice, we found that homozygous syntaxin-1AKO 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-1BOpen 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-1BOpen mutant mice was recessive and independent of the syntaxin-1AKO. Thus, syntaxin-1B was selectively essential, probably because it is more widely expressed than syntaxin-1A (15). In C. elegans, transgenic syntaxin-1Open rescues the paralysis of unc-13 mutant worms (16), but crossing syntaxin-1BOpen mice with Munc13-1 knockout mice did not rescue the Munc13-1 knockout-induced lethality (Fig. S4).
The syntaxin-1AKO mutation abolished syntaxin-1A expression (Fig. 1B), whereas the syntaxin-1BOpen 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-1Open 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-1BOpen 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-1BOpen and -1BWT mice lacking syntaxin-1A revealed increased vesicle docking in syntaxin-1BOpen 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-1BOpen and -1BWT synapses; in particular, the number and intraterminal distribution of vesicles were unaltered (Fig. S7). In chromaffin cells, however, the syntaxin-1BOpen 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-1AKO and wild-type synapses (12). In syntaxin-1BOpen synapses (on the syntaxin-1AKO 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-1BOpen 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-1AKO synapses, but decreased ~35% in syntaxin-1BOpen synapses (Figs. 4A and 4B), consistent with the decreased syntaxin-1 and Munc18-1 levels in syntaxin-1BOpen 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 Pvr (as the ratio of EPSC and RRP charges). Both were increased >2-fold in syntaxin-1BOpen synapses (Figs. 4C and 4D), augmenting the percentage of the RRP released by a single action potential from ~10% in syntaxin-1BWT to ~20% in syntaxin-1BOpen 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-1BOpen 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-1BOpen 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-1BOpen 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-1BOpen mutant mice, we hypothesize that the syntaxin-1BOpen 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-1BOpen 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 ). 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.
We thank Ms. N. Hamlin, I. Kornblum, A. Roth, I. Herfort, and E. Borowicz for technical assistance. This work was supported by grants from the DFG (GE1042/1-1, GE1042/3-1, GE1042/3-2 to S.H.G.; Ro1296/5-3 to C.R.), the NIH (NS051262 to C.R., NS37200 to J.R.), and the NOSR (GpD970-10-036 and 900-01-001 to MV; 916-36-043 to HdW).