A. and B. Representative electron micrographs of neurons cultured from syntaxin-1A^KO mice containing wild-type syntaxin-1B^WT (A) or ‘open’ syntaxin-1B^Open (B; scale bar, 250 nm).
C. Number of docked vesicles per active zone (n = 3 experiments with 1B^WT=49, 55, and 32 synapses; 1B^Open=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: p<0.01; 1B^WT=136, 1B^Open=108 synapses).
E. and F. Size of the postsynaptic density (E) and number of docked vesicles/length of postsynaptic density (F; both normalized for wild-type values).
G. - J. Representative electron micrographs of chromaffin cells from control (G,H) and syntaxin-1B^Open (I,J) littermate mice at embryonic day E18 at two magnifications (G,I: bar, 1 μm; H,J: bar, 200 nm).
K. Distribution of the distance of secretory granules from the plasma membrane in chromaffin cells from syntaxin-1B^WT, -1B^Open, Munc18-1 KO, and synaptobrevin-2 KO mice (analyzed separately with wildtype controls and binned as indicated).
L. Total number of secretory granules per chromaffin cell in syntaxin-1B^WT or -1B^Open 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 to wild-type.

A. Average postsynaptic currents elicited by application of 0.5 M sucrose in syntaxin-1B^WT and -1B^Open 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 (mini frequency divided by the number of vesicles in the RRP)
D. Mean vesicular release probability (P_vr = evoked EPSC charge divided by the RRP charge).
E. Time-course of the average cumulative synaptic charge transfer during sucrose-induced release. For syntaxin-1B^Open 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 vs. time-to-onset of sucrose-induced synaptic responses.
G. Representative traces of synaptic responses induced by 0.25 M, 0.35 M 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 to wild-type; numbers in bars show numbers of neurons analyzed.

A. Survival of syntaxin-1 mutant mice
B. and C. Representative immunoblots (B) and levels of synaptic proteins (C) from syntaxin-1A^WT/1B^WT, -1A^WT/1B^Open, -1A^KO/1B^WT, and -1A^KO/1B^Open mutant mice determined by quantitative immunoblotting using ¹²⁵I-labeled secondary antibodies (see also Table S1).
D. Representative immunoblots (left) and quantitations (right) analyzing co-immunoprecipitation of Munc18-1, SNAP-25, synaptobrevin-2, and synaptotagmin-1 with syntaxin-1B^WT and -1B^Open in Triton X-100 solubilized brain proteins. The amounts of co-immunoprecipitated 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 to wild-type. Abbreviations: Synt., syntaxin; Syb-2, synaptobrevin-2; Synph-1, synaptophysin-1; Syt-1, synaptotagmin-1; VCP, p97/vasolin-containing protein; GDI, GDP dissociation inhibitor.

A.- C. Summary graphs of the frequency (A), amplitude (B), and charge (C) of spontaneous synaptic events (mEPSCs)
D. - F. Representative traces (D) and 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 stimulus trains (H).
Data are means ± SEMs; *=p<0.05 by Student's t-test compared to wild-type; numbers in bars show numbers of neurons analyzed.