Fig 2: ‘Opening’ of STX1A in combination with the deletion of its entire N-terminal stretch does not impair neurotransmitter release.(A) Example images of immunofluorescence labeling for Bassoon, STX1A, and Munc18-1 shown as red, green, and blue, respectively, in the corresponding composite pseudocolored images obtained from high-density cultures of STX1-null hippocampal neurons either not rescued or rescued with STX1AWT, STX1ALEOpen, STX1ALEOpen + ?N2-9, STX1ALEOpen + ?N2-19, or STX1ALEOpen + ?N2-28. Scale bar: 10 µm (B, C) Quantification of the immunofluorescence intensity of STX1A and Munc18-1 as normalized to the immunofluorescence intensity of Bassoon in the same ROIs as shown in (A). The values were then normalized to the values obtained from STX1AWT neurons. (D) Example traces (left) and quantification of the amplitude (right) of EPSCs obtained from hippocampal autaptic STX1AWT, STX1ALEOpen, STX1ALEOpen + ?N2-9, STX1ALEOpen + ?N2-19, or STX1ALEOpen + ?N2-28 neurons. (E) Example traces (left) and quantification of the charge transfer (right) of sucrose-elicited readily releasable pools (RRPs) obtained from the same neurons as in (D). (F) Quantification of probability of vesicular release (Pvr) determined as the percentage of the RRP released upon one action potential (AP). (G) Example traces (left) and quantification (right) of paired-pulse ratio (PPR) measured at 40 Hz. (H) Example traces (left) and quantification of the frequency (right) of mEPSCs. (I) Quantification of mEPSC rate as spontaneous release of one unit of RRP. (I) Quantification of mEPSC rate as spontaneous release of one unit of RRP. Figure 4—source data 1.Quantification of lentiviral expression of STX1ALEOpen and STX1ALEOpen + ?? mutants in STX1-null neurons and the consequent neurotransmitter release properties.

Fig 3: STX1A’s Habc-domain is essential and N-peptide is dispensable for neurotransmitter release.(A) Domain structure of STX1A. The protein consists of a short N-peptide (aa 1–9 or 1–28), Habc domain (aa 29–144) formed by three helices, Ha, Hb, and Hc, followed by the H3 helix (aa 189–259; SNARE domain) and a transmembrane region (aa 266–288; TMR). (B) Example images of immunofluorescence labeling for Bassoon, STX1A, and Munc18-1 shown as red, green, and blue, respectively, in the corresponding composite pseudocolored images obtained from high-density cultures of STX1-null hippocampal neurons either not rescued or rescued with STX1AWT, or STX1A?2-9; STX1ALEOpen; or STX1A?Habc. Scale bar: 10 µm (C, D) Quantification of the immunofluorescence intensity of STX1A and Munc18-1 as normalized to the immunofluorescence intensity of Bassoon in the same ROIs as shown in (B). The values were then normalized to the values obtained from STX1AWT neurons. (E) Example traces (left) and quantification of the amplitude (right) of EPSCs obtained from hippocampal autaptic STX1-null neurons either not rescued or rescued with STX1AWT, STX1B?2-9, STX1ALEOpen, or STX1A?Habc. (F) Example traces (left) and quantification of the charge transfer (right) of 500 mM sucrose-elicited readily releasable pools (RRPs) obtained from the same neurons as in (E). (G) Quantification of probability of vesicular release (Pvr) determined as the percentage of the RRP released upon one AP. (H) Example traces (left) and quantification of the frequency (right) of mEPSCs recorded at –70 mV. (I) Example traces (left) and quantification (right) of short-term plasticity (STP) determined by high-frequency stimulation at 10 Hz and normalized to the EPSC1 from the same neuron. Data information: the artifacts are blanked in example traces in (D) and (H). The example traces in (G) were filtered at 1 kHz. In (C–H), data points represent single observations, the bars represent the mean ± SEM. In (I), data points represent mean ± SEM. Red and black annotations (stars and n.s.) on the graphs show the significance comparisons to STX1-null and to STX1AWT rescue, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p=0.05, ***p=0.001, ****p=0.0001). Two-way ANOVA was applied for data in (I). The numerical values are summarized in Figure 1—source data 1. Figure 1—source data 1.Quantification of the STX1AWT and mutant STX1A expression induced by lentiviral transduction of STX1-null neurons and the consequent neurotransmitter release properties.

Fig 4: The Juxtamembrane Motif of the v-SNARE Is Embedded in the Surface of the Lipid Bilayer(A) Sequence alignment of the juxtamembrane motifs (highlighted in yellow) of exocytic v-SNAREs from multiple species. The SNARE motif is also known as the core domain. TMD, transmembrane domain.(B) Model of the VAMP2 juxtamembrane motif embedded in a membrane bilayer. Magenta, VAMP2 (v-SNARE, the juxtamembrane motif is highlighted in yellow); green, syntaxin-1 (t-SNARE); blue, SNAP-25 (t-SNARE, only the SNARE motifs are shown); orange, Munc18-1 (SM protein, shown as a surface model). The juxtamembrane motif of VAMP2 is based on previous biophysical and structural data (Bowen and Brunger, 2006; Brewer et al., 2011; Ellena et al., 2009; Kweon et al., 2003). The TMD of VAMP2 is tilted about 35° relative to the membrane normal to allow the nonpolar residues of the juxtamembrane motif to insert into the hydrophobic phase of the bilayer and the basic residues to embed in the hydrophilic phase of the membrane. Carbon, oxygen, and phosphorus atoms of the lipid bilayer are colored gray, red, and green, respectively. The model is based on the structures of the v-SNARE (PDB: 2KOG; Ellena et al., 2009), the cis-SNARE complex (PDB: 3HD7; Stein et al., 2009), and Munc18-1 (PDB: 3PUJ; Hu et al., 2011). Because the structure of Munc18-1 bound to the half-zippered trans-SNARE complex has not been determined, the position of Munc18-1 depicted in the model is arbitrary. The model of the phosphatidylcholine bilayer (popc128a.pdb) was obtained from the Department of Biocomputing at the University of Calgary (Calgary, AB, Canada). The models were prepared using PyMOL (DeLano Scientific LLC, San Carlos, CA).

Fig 5: The Juxtamembrane Motif of VAMP2 Is Essential for SNARE-Munc18-1-Mediated Membrane Fusion(A) Sequence alignment of the juxtamembrane motifs of WT VAMP2 and a VAMP2 mutant in which the juxtamembrane motif was mutated into alanines. Asterisks indicate the conserved residues mutated in the VAMP2 mutant. Lysine 94 (K94) was not mutated because this basic residue demarcates the boundary of the TMD. Lysine 91 (K91) was not mutated because it is not evolutionarily conserved. Nevertheless, identical results were observed when K91 was also mutated (Figure 6).(B) Representative Coomassie blue-stained gel showing that WT and mutant VAMP2 proteins were reconstituted into proteoliposomes at comparable levels.(C) Diagram of liposome pairs in the reconstituted liposome fusion reactions. WT t-SNARE liposomes were directed to fuse with v-SNARE liposomes containing WT or mutant VAMP2 in the absence or presence of 5 µM Munc18-1.(D) Lipid mixing of the liposome fusion reactions. In negative control reactions, the dominant-negative inhibitor CDV2 (cytoplasmic domain of VAMP2) was added to a final concentration of 20 µM. Content mixing of the liposomes is shown in Figure S2.(E) Initial lipid-mixing rates of the liposome fusion reactions in (D). Data are presented as average percentage of fluorescence change within the initial 10 min of the reactions based on three independent experiments. Error bars indicate SD.See also Figures S1 and S2.