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Science 321 (5895): 1507-1510

Copyright © 2008 by the American Association for the Advancement of Science

Conformational Switch of Syntaxin-1 Controls Synaptic Vesicle Fusion

Stefan H. Gerber1*{dagger}, Jong-Cheol Rah2,3*{ddagger}, Sang-Won Min1*§, Xinran Liu1,4, Heidi de Wit5, Irina Dulubova6, Alexander C. Meyer3, Josep Rizo6,7, Marife Arancillo2, Robert E. Hammer6,7, Matthijs Verhage5, Christian Rosenmund2,3#, and Thomas C. Südhof1,4,8#

1 Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390–9111, USA.
2 Department of Molecular and Human Genetics and Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
3 Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
4 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390–9111, USA.
5 Department of Functional Genomics, Vrije Universiteit, 1081 Amsterdam, Netherlands.
6 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390–9111, USA.
7 Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390–9111, USA.
8 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390–9111, USA.


Figure 1 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. [View Larger Version of this Image (46K GIF file)]
 

Figure 2 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. [View Larger Version of this Image (89K GIF file)]
 

Figure 3 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. [View Larger Version of this Image (29K GIF file)]
 

Figure 4 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. [View Larger Version of this Image (35K GIF file)]
 


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