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Conformational Switch of Syntaxin-1 Controls Synaptic Vesicle Fusion

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Science  12 Sep 2008:
Vol. 321, Issue 5895, pp. 1507-1510
DOI: 10.1126/science.1163174

Abstract

During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor–attachment protein receptor (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. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1BOpen mice were viable but succumbed to generalized seizures at 2 to 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; however, 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 [soluble N-ethylmaleimide–sensitive factor (NSF)–attachment protein receptor) and SM (Sec1-Munc18–like) proteins (1, 2). In Ca2+-triggered exocytosis in neurons and neuroendocrine cells, fusion is catalyzed by the formation of SNARE complexes from syntaxin-1, synaptosome-associated protein of 25 kDa (SNAP-25), and synaptobrevin/vesicle-associated membrane protein and the binding of the SM protein Munc18-1 to these SNARE complexes (13). 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 Munc18-1–SNARE complex assemblies (7, 8), which are essential for exocytosis (3). The function 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) (9). 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 (1014) and indicated that syntaxin-1A may be functionally redundant with syntaxin-1B.

Fig. 1.

Syntaxin-1AKO/syntaxin-1BOpen double mutant mice perish postnatally. (A) Survival of syntaxin-1 mutant mice. Numbers in brackets show the total number of analyzed mice. Representative immunoblots (B) and levels of synaptic proteins (C) from syntaxin-1AWT/1BWT, -1AWT/1BOpen, -1AKO/1BWT, and -1AKO/1BOpen mutant mice determined by quantitative immunoblotting with 125I-labeled secondary antibodies (table S1). *, P < 0.05. (D) Representative immunoblots (left) and quantitations (right) analyzing coimmunoprecipitation of Munc18-1, SNAP-25, synaptobrevin-2, and synaptotagmin-1 with syntaxin-1BWT and -1BOpen in brain proteins solubilized with Triton X-100 (St. Louis, MO). The amounts of coimmunoprecipitated Munc18-1, SNAP-25, synaptobrevin-2, and synaptotagmin-1 were determined by quantitative immunoblotting and normalized for the immunoprecipitated syntaxin-1. Data in (C) and (D) are means ± SEMs; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t test compared with wild type. Synt, syntaxin; Syb-2, synaptobrevin-2; Synph-1, synaptophysin-1; Syt-1, synaptotagmin-1; VCP, p97/vasolin-containing protein; GDI, guanosine diphosphate dissociation inhibitor.

Homozygous mutant syntaxin-1BOpen mice were also viable but severely ataxic and developed lethal epileptic seizures after 2 weeks of age (Fig. 1A and fig. 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 Caenorhabditis elegans, transgenic syntaxin-1Open rescues unc-13 mutant worms from paralysis (16); however, crossing syntaxin-1BOpen mice with Munc13-1 knockout mice did not prevent Munc13-1 knockout–induced death (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 (fig. S5) (3, 8). Consistent with this conclusion, less Munc18-1 was coimmunoprecipitated with syntaxin-1 in syntaxin-1BOpen mice, whereas other SNARE proteins coimmunoprecipitated normally (Munc18-1–SNARE complex assemblies are not stable during immunoprecipitations, and thus cannot be evaluated) (Fig. 1D and fig. S6).

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) (Fig. 2, A to D). The size of the postsynaptic density also was increased (∼20%) (Fig. 2E), whereas 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 that of the Munc18-1 knockout. Again, neither mutation altered the total number of chromaffin vesicles (Fig. 2, K and L). Synaptobrevin-2 knockout mice, analyzed in parallel as a negative control, did not change chromaffin vesicle docking but did increase the total number of chromaffin vesicles (Fig. 2L). Consistent with earlier findings (1720), 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.

Fig. 2.

Syntaxin-1BOpen impairs chromaffin but not synaptic vesicle docking. (A and B) Representative electron micrographs of neurons cultured from syntaxin-1AKO mice containing wild-type syntaxin-1BWT (A) or open syntaxin-1BOpen (B) (scale bar, 250 nm). (C) Number of docked vesicles per active zone (n = 3 experiments performed with 1BWT = 49, 55, and 32 synapses and 1BOpen = 21, 38, and 49 synapses; normalized for wild-type values). (D) Plot of the cumulative distribution of docked vesicles per active zone (statistical significance with Kolmogorov-Simirnov test is P < 0.01; 1BWT = 136 synapses and 1BOpen = 108 synapses). (E and F) Size of the postsynaptic density (PSD) (E) and number of docked vesicles/length of postsynaptic density (F) (both normalized for wild-type values). (G to J) Representative electron micrographs of chromaffin cells from control [(G) and (H)] and syntaxin-1BOpen [(I) and (J)] littermate mice at embryonic day E18 at two magnifications [(G) and (I), scale bar indicates 1 μm; (H) and (J), scale bar indicates 200 nm). (K) Distribution of the distance of secretory granules from the plasma membrane in chromaffin cells from syntaxin-1BWT, -1BOpen, Munc18-1 knockout, and synaptobrevin-2 knockout mice (analyzed separately with wild-type controls and binned as indicated). (L) Total number of secretory granules per chromaffin cell in syntaxin-1BWT or -1BOpen mice and in Munc18-1 and synaptobrevin-2 KO mice [(K) and (L), N = 3 animals, n = 60 chromaffin cells]. Data are means ± SEMs; **, P < 0.01 by Student's t test compared with wild type.

Measurements of spontaneous miniature excitatory postsynaptic currents (mEPSCs), excitatory postsynaptic currents (EPSCs) evoked by isolated action potentials, and use-dependent synaptic depression during high-frequency stimulus trains in hippocampal neurons revealed no significant difference between syntaxin-1AKO and wild-type (WT) 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 evoked EPSCs unexpectedly exhibited normal amplitude and kinetics (Fig. 3 and fig. S8).

Fig. 3.

Neurotransmitter release in syntaxin-1BOpen synapses. (A to C) Summary graphs of the frequency (A), amplitude (B), and charge (C) of spontaneous mEPSCs. (D to F) Representative traces (D), mean EPSC amplitudes (E), and charges (F) in synaptic responses induced by isolated action potentials. (G and H) EPSC amplitudes of evoked synaptic responses elicited by 10 Hz (G) and 20 Hz (H) stimulus trains. Data are means ± SEMs; *, P < 0.05 by Student's t test compared with wild type. Numbers in bars show numbers of neurons analyzed.

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 these possibilities, we measured the RRP by applying 0.5 M sucrose (9, 21). The RRP was unchanged in syntaxin-1AKO synapses but decreased ∼35% in syntaxin-1BOpen synapses (Fig. 4, A and B), which is consistent with 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 (Fig. 3, A to F) 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 more than two-fold in syntaxin-1BOpen synapses (Fig. 4, C and D), 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, not a decrease (fig. S9).

Fig. 4.

Increased fusogenicity of synaptic vesicles in syntaxin-1BOpen synapses. (A) Average postsynaptic currents elicited by application of 0.5 M sucrose in syntaxin-1BWT and -1BOpen synapses. (B) Mean RRP size determined as the transient charge integral induced by application of 0.5 M hypertonic solution. (C) Summary graph of the spontaneous vesicular release rate (minifrequency divided by the number of vesicles in the RRP). (D) Mean Pvr. (E) Time course of the average cumulative synaptic charge transfer during sucrose-induced release. For syntaxin-1BOpen synapses, both absolute (blue, left y axis) and normalized responses (red, right y axis) are depicted. In (B) and (E), the steady-state component of release during the responses was subtracted. (F) Plot of the half-width versus the time-to-onset of sucrose-induced synaptic responses. (G) Representative traces of synaptic responses induced by 0.25, 0.35, and 0.50 M sucrose. (H and I) Plot of the released fraction of the RRP [(H) defined as the response to 0.5 M sucrose] and of the vesicular release rate (I) as a function of the sucrose concentration. In (G), the 0 mM sucrose value represents the spontaneous vesicular release rate (Fig. 4D). Data are means ±SEMs; **, P < 0.01; ***, P < 0.001 by Student's t test compared with wild type. Numbers in bars show numbers of neurons analyzed.

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 (Fig. 4, E and F) and significantly boosts the relative amount and fractional release rate induced at lower sucrose concentrations (Fig. 4, G to I). 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.

The closed conformation of syntaxin-1 performs three functions upstream of the canonical role of syntaxin-1 as a SNARE protein in membrane fusion: (i) Closed syntaxin-1, but not the SNARE complex, mediates vesicle docking in chromaffin cells but not in synapses. The same differential phenotype is observed upon deletion of Munc18-1 (17, 18), suggesting that the Munc18-1–syntaxin-1 complex docks chromaffin but not synaptic vesicles (fig. S12). (ii) The closed syntaxin-1 conformation stabilizes syntaxin-1 and Munc18-1, whereas opening syntaxin-1 decreases syntaxin-1 and Munc18-1 levels and thereby lowers the RRP size. (iii) Opening syntaxin-1 accelerates the rate of synaptic vesicle fusion, accounting for the fulminant epilepsy observed in synaxin-1BOpen mutant mice.

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 (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 over 100-fold less 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.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1163174/DC1

Materials and Methods

Figs S1 to S13

Table S1 and S2

References

References and Notes

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