Reexamination of N-terminal domains of syntaxin-1 in vesicle fusion from central murine synapses

Abstract

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX1's closed conformation driven by its H_abc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, H_abc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the H_abc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca²⁺-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.

Keywords: Munc18-1; N-peptide; habc-domain; leopen conformation; mouse; neuroscience; synaptic transmission; syntaxin-1.

Figure 1.. STX1A’s H_abc-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), H_abc domain (aa 29–144) formed by three helices, H_a, H_b, and H_c, 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 STX1A^WT, or STX1A^∆2-9; STX1A^LEOpen; 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 STX1A^WT 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 STX1A^WT, STX1B^∆2-9, STX1A^LEOpen, 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 STX1A^WT 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—figure supplement 1.. STX1A^∆Habc expression cannot be detected.

(A) Example images (left) and quantification of immunofluorescence labeling for Bassoon and STX1A shown as red and green, respectively, in the corresponding composite pseudocolored images obtained from high-density cultures of STX1-null hippocampal neurons either not rescued or rescued with STX1A^WT or STX1A^WT-FLAG. The data were normalized to the STX1A fluorescence/Bassoon fluorescence value obtained from STX1A^WT neurons. Scale bar: 10 µm (B) Example images (left) and quantification of immunofluorescence labeling for Bassoon and FLAG shown as red and green, respectively, in the corresponding composite pseudocolored images obtained from high-density cultures of STX1-null hippocampal neurons rescued with either STX1A^WT, STX1A^WT-FLAG, STX1A^∆N2-9-FLAG, STX1A^LEOpen-FLAG, or STX1A^∆Habc-FLAG. The data were normalized to the STX1A fluorescence/Bassoon fluorescence value obtained from STX1A^WT-FLAG neurons. Scale bar: 10 µm Data information: data points in graphs represent single observations, and the bars represent the mean ± SEM. In (A), black and red annotations on the graphs show the significance comparisons to STX1-null and STX1A^WT, respectively. In (B), black and red annotations on the graphs show the significance comparisons to STX1^WT and STX1A^WT-FLAG, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 1—figure supplement 1—source data 1.

Figure 2.. STX1’s H_abc-domain is essential for the overall function of STX1A.

(A) Example images of high-density cultures of STX1-null, STX1A^WT, and STX1A^∆Habc hippocampal neurons at DIV 8, 15, 22, and 29 represented with immunofluorescent labeling of microtubule associated protein 2 (MAP2) . Red and green nuclei serve as a marker for NLS-RFP-P2A-Cre recombinase expression and for NLS-GFP-P2A-STX1A (either WT or mutants), respectively. Scale bar: 50 µm. (B) Quantification of neuronal density at DIV 8. (C) Quantification of the percentage of the surviving neurons at DIV 8, 15, 22, and 29 as normalized to the neuronal density at DIV 8 in the same well. (D) Example high-pressure freezing fixation combined with electron microscopy (HPF-EM) images of nerve terminals from high-density cultures of STX1-null hippocampal neurons either not rescued or rescued with STX1A^WT or STX1A^∆Habc. (E–G) Quantification of active zone (AZ) length, number of synaptic vesicles (SVs) within 200 nm distance from AZ, and number of docked SVs. (H, I) SV distribution of STX1-null and STX1A^∆Habc neurons compared to that of STX1A^WT neurons. Data information: in (B, E–G), data points represent single observations, the bars represent the mean ± SEM. In (C, H, I), data points represent the mean ± SEM. Red and black annotations (stars and n.s.) on the graphs show the significance comparisons to STX1-null and to STX1A^WT neurons, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 2—source data 1.

Figure 3.. Deletion of the 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 STX1A^WT, STX1A^∆2-9, STX1A^∆2-19, or STX1A^∆2-28. 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 STX1A^WT neurons. (D) Example traces (left) and quantification of the amplitude (right) of EPSCs obtained from hippocampal autaptic STX1-null neurons either not rescued or rescued with STX1A^WT, STX1A^∆2-9, STX1A^∆2-19, or STX1A^∆2-28. (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. The artifacts are blanked in the example traces. (H) Example traces (left) and quantification of the frequency (right) of mEPSCs. The example traces were filtered at 1 kHz. (I) Quantification of mEPSC rate as spontaneous release of one unit of RRP. Data information: the artifacts are blanked in example traces in (D) and (G). The example traces in (H) were filtered at 1 kHz. In (B–I), data points represent single observations, the bars represent the mean ± SEM. Red and black annotations (stars and n.s.) on the graphs show the significance comparisons to STX1-null and to STX1A^WT neurons, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, ****p≤0.0001). The numerical values are summarized in Figure 3—source data 1.

Figure 3—figure supplement 1.. Phosphorylation of S14 of STX1 has no effect on synaptic transmission.

(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, STX1A^WT, STX1A^S14A, or STX1A^S14E neurons. 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 STX1-null neurons expressing STX1A^WT. (D) Example traces (left) and quantification of the amplitude (right) of EPSCs obtained from hippocampal autaptic STX1-null neurons either not rescued or rescued STX1A^WT, or STX1B^S14A, or STX1A^S14E. (E) Example traces (left) and quantification of the charge transfer (right) of sucrose-elicited readily releasable pools (RRPs). (F) Quantification of probability of vesicular release (Pvr) determined as the percentage of the RRP released upon one action potential (AP). (G) Quantification of paired-pulse ratio (PPR) of EPSCs elicited at 40 Hz. (H) Example traces (left) and quantification of the frequency (right) of mEPSCs recorded at –70 mV. The example traces were filtered at 1 kHz. (I) Example traces (left) and quantification (right) of 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 In (D) and (I). In (B–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 STX1A^WT rescue, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p≤0.05, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 3—figure supplement 1—source data 1.

Figure 3—figure supplement 2.. Modifications of STX1’s N-peptide either by deletions or phosphorylation does not compromise neuronal viability.

(A) Example images of high-density cultures of STX1-null hippocampal neurons at DIV 8 and 29 represented with immunofluorescent labeling of microtubule associated protein 2 (MAP2). Red and green nuclei serve as a marker for NLS-RFP-P2A-Cre recombinase expression and for NLS-GFP-P2A-STX1A (either WT or mutants), respectively. Scale bar: 50 µm. (B) Quantification of neuronal density at DIV 8 in STX1-null hippocampal neurons either not rescued or rescued with STX1A^WT or STX1A^∆N2-9, STX1A^∆N2-19, STX1A^∆N2-28, STX1A^S14A, or STX1A^S14E. (B) Quantification of the percentage of the surviving neurons at DIV 29 as normalized to the neuronal density at DIV 8. Data information: in (B, C), data points represent single observations, the bars represent the mean ± SEM. Red and black annotations (stars and n.s.) on the graphs show the significance comparisons to STX1-null and to STX1A^WT rescue, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, **p≤0.01, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 3—figure supplement 2—source data 1.

Figure 4.. ‘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 STX1A^WT, STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆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 STX1A^WT neurons. (D) Example traces (left) and quantification of the amplitude (right) of EPSCs obtained from hippocampal autaptic STX1A^WT, STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆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—figure supplement 1.. Interruption of both Munc18-1 binding modes of STX1 ultimately leads to neuronal death.

(A) Example images of high-density cultures of STX1-null hippocampal neurons at DIV 8, 15, 22, 29, 36, and 43 represented with immunofluorescent labeling of microtubule associated protein 2 (MAP2). Red and green nuclei serve as a marker for NLS-RFP-P2A-Cre recombinase expression and for NLS-GFP-P2A-STX1A (either WT or mutants), respectively. 50 µm. (A) Quantification of neuronal density at DIV 8 in STX1-null hippocampal neurons either not rescued or rescued with STX1A^WT, STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆N2-28}. Scale bar: 10 µm (B) Quantification of the percentage of the surviving neurons at DIV 8, 15, 22, 29, 36, and 43 as normalized to the neuronal density at DIV 8. Data information: in (B), data points represent single observations, the bars represent the mean ± SEM. In (C), data points represent the mean ± SEM. Red and black annotations (stars and n.s.) on the graphs show the significance comparisons to STX1-null and to STX1A^WT rescue, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). The number of observations and cultures and statistical data are summarized in Figure 4—source data 1. The numerical values are summarized in Figure 4—figure supplement 1—source data 1.

Figure 4—figure supplement 2.. Reducing the expression levels of STX1A^WT or STX1A^LEOpen does not alter their synaptic release properties.

(A) Example images of immunofluorescence labeling for VGlut1, STX1A, Munc18-1, and NLS-GFP shown as red, green, blue, and gray, respectively, in autaptic STX1-null hippocampal neurons rescued with 1× viral volume of STX1A^{LEOpen + ∆N2-28} as reference or with different viral volumes of either STX1A^WT, or STX1A^LEOpen as. Scale bar: 50 µm. (A) Higher magnification images of the areas highlighted in the images in (A). VGlut1, STX1A, and Munc18-1 are shown as red, green, and blue, respectively, in the corresponding composite image. Scale bar: 10 µm. (B) Quantification of the fluorescence intensity of NLS-GFP from the neurons as in (A) as normalized to that of STX1A^WT 1× rescue. (C) Quantification of the immunofluorescence intensity of STX1A 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 STX1A^WT 1× neurons. (D) Quantification of the immunofluorescence intensity of 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 STX1-null neurons expressing STX1A^WT 1× neurons. Example traces (left) and quantification of the amplitude (right) of EPSCs obtained from hippocampal autaptic STX1-null neurons rescued with 1× viral volume of STX1A^{LEOpen + ∆N2-28} as reference or with different viral volumes of either STX1A^WT, or STX1A^LEOpen. The artifacts are blanked in the example traces. (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 (F), (G) Quantification of probability of vesicular release (Pvr) determined as the percentage of the RRP released upon one action potential. (H) Example traces (left) and quantification of the frequency (right) of mEPSCs recorded at –70 mV. The example traces were filtered at 1 kHz. Data information: in (C–I), data points represent single observations, the bars represent the mean ± SEM. Red annotations (stars and n.s.) on the graphs show the significance comparisons to STX1A^{LEOpen + ∆N2-28} 1× rescue. Black annotations (stars and n.s.) on the bars show the significance comparisons for rescues using different viral volume either for STX1A^WT or STX1A^LEOpen. (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 4—figure supplement 2—source data 1.

Figure 4—figure supplement 3.. Exogenous expression of STX1A using 1× volume of lentiviral particles is approximately threefold higher than endogenous STX1A expression.

(A) Example images of immunofluorescence labeling for VGlut1, STX1A, and Munc18-1. Scale bar: 10 µm. (B) Quantification of the immunofluorescence intensity of STX1A 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 WT neurons. (C) Quantification of the immunofluorescence intensity of 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 WT neurons. Data information: in (B, C), data points represent single observations, the bars represent the mean ± SEM. Red annotations (stars and n.s.) on the graphs show the significance comparisons of endogenous STX1A vs. exogenous STX1A using 1× viral volume. (Mann–Whitney test, ***p≤0.001). The numerical values are summarized in Figure 4—figure supplement 3—source data 1.

Figure 5.. ‘Opening’ of STX1A in combination with the deletion of its entire N-terminal stretch reduces the number of docked synaptic vesicles (Svs).

(A) Example high-pressure freezing fixation combined with electron microscopy (HPF-EM) images of nerve terminals from high-density cultures of STX1A^WT, STX1A^∆N2-28, STX1A^LEOpen, and STX1A^{LEOpen + ∆N2-28} Neurons. (B–D) Quantification of active zone (AZ) length, number of SVs within 200 nm distance from AZ, and number of docked SVs. (E) Correlation of the number of docked SVs obtained by HPF-EM to the size of readily releasable pool (RRP) obtained by electrophysiological recordings. (F–H) SV distribution of STX1A^∆N2-28, STX1A^LEOpen, and STX1A^{LEOpen + ∆N2-28} neurons compared to that of STX1A^WT neurons. Data information: in (B–D), data points represent single observations, the bars represent the mean ± SEM. In (E–H), data points represent mean ± SEM. Black annotations on the graphs show the significance comparisons to STX1A^WT rescue (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test in B–D, multiple t-tests in F–H *p≤0.05, **p≤0.01, ***p≤0.001). The numerical values are summarized in Figure 5—source data 1.

Figure 5—figure supplement 1.. Synapse number and area are not affected neither by LE_Open mutation nor by LE_Open + ΔN mutation.

(A) Quantification of VGlut1-positive puncta using the images exemplified in Figure 4—figure supplement 2 and obtained from STX1A^WT, STX1A^LEOpen, and STX1A^{LEOpen + ∆N2-28} neurons. (B) Quantification of the total area of VGlut1-positive puncta using the images exemplified in Figure 4—figure supplement 2 and obtained from STX1A^WT, STX1A^LEOpen and STX1A^{LEOpen + ∆N2-28} neurons. Data information: data points represent single observations; the bars represent the mean ± SEM. Red annotations (n.s.) on the graphs show the significance comparisons to comparisons to STX1A^WT (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test). The numerical values are summarized in Figure 5—figure supplement 1—source data 1.

Figure 6.. STX1A’s N-peptide has a modulatory function in short-term plasticity and Ca²⁺-sensitivity of synaptic transmission.

(A) Example traces (left) and quantification (right) of STP measured by 50 stimulations at 10 Hz from STX1A^WT, STX1A^∆N2-9, STX1A^∆N2-19, or STX1A^∆N2-28 neurons. The traces show the absolute values, whereas the quantification shows normalized EPSC to EPSC₁. (B) Example traces (left) and quantification (right) of the recovery of readily releasable pool (RRP) determined as the fraction of RRP measured at a second pulse of 500 mM sucrose solution after 2 s of initial depletion from STX1A^WT, STX1A^∆N2-9, STX1A^∆N2-19, or STX1A^∆N2-28 neurons. (C) Example traces (left) and quantification (right) of the ratio of the charge transfer triggered by 250 mM sucrose over that of 500 mM sucrose as a read-out of fusogenicity of the synaptic vesicles (SVs). (D) Example traces (left) and quantification (right) of Ca²⁺-sensitivity as measured by the ratio of EPSC amplitudes at [Ca²⁺]_e of 0.5, 1, 2, 4, and 10 mM recorded from STX1A^WT, STX1A^∆N2-9, or STX1A^∆N2-28 neurons. The responses were normalized to the response at [Ca²⁺]_e of 10 mM. (E) Paired-pulse ratio (PPR) of EPSC amplitudes at [Ca²⁺]_e of 0.5, 1, 2, 4, and 10 mM recorded at 40 Hz. (F) Example traces (left) and quantification (right) of STP measured by 50 stimulations at 10 Hz from STX1A^WT, STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆N2-28} neurons. The traces show the absolute values, whereas the quantification shows normalized EPSC to EPSC₁. (G) Example traces (left) and quantification (right) of the recovery of RRP determined as the fraction of RRP measured at a second pulse of 500 mM sucrose solution after 2 s of initial depletion from STX1A^WT, STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆N2-28} neurons. (H) Example traces (left) and quantification (right) of the ratio of the charge transfer triggered by 250 mM sucrose over that of 500 mM sucrose as a read-out of fusogenicity of the SVs. (I) Example traces (left) and quantification (right) of Ca²⁺-sensitivity recorded from STX1A^WT, STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, or STX1A^{LEOpen + ∆N2-28} neurons. The responses were normalized to the response at [Ca²⁺]_e of 10 mM. (J) PPR of EPSC amplitudes at [Ca²⁺]_e of 0.5, 1, 2, 4, and 10 mM recorded at 40 Hz from STX1A^WT, STX1A^LEOpen, or STX1A^{LEOpen + ∆N2-28} neurons. Data information: the artifacts are blanked in example traces in (A, D, F, I). In (A, D, E, F, I, J), data points represent the mean ± SEM. In (B, C, G, H), data points represent single observations, the bars represent the mean ± SEM. Black and red annotations on the graphs show the significance comparisons to STX1A^WT or STX1A^LEOpen, respectively. (either nonparametric Kruskal–Wallis followed by Dunn’s post hoc test or one-way ANOVA followed by Holm–Sidak’s post hoc test was applied based on the normality of the data, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 6—source data 1.

Figure 6—figure supplement 1.. Deletion of N-peptide increases the paired-pulse ratio (PPR) both in closed and open conformation of STX1A in low extracellular Ca²⁺-concentration.

(A) Quantification of absolute values of 50 EPSCs elicited at 10 Hz recorded from neurons expressing either STX1A^WT, STX1A^∆N2-9, STX1A^∆N2-19, or STX1A^∆N2-28. (B) Example traces (left) and quantification (right) of Ca²⁺-sensitivity recorded from neurons expressing STX1A^∆N2-19, compared to that of neurons expressing STX1A^WT. The responses were normalized to the response at [Ca²⁺]_e of 10 mM. The artifacts are blanked in the example traces. (C) Example traces (left) and quantifications (right) of STP measured at [Ca²⁺]_e of either 0.5 mM, 2 mM, or 10 mM by five stimulations at 40 Hz from neurons expressing either STX1A^WT, STX1A^∆N2-9, or STX1A^∆N2-28. The artifacts are blanked in the example traces. At every [Ca²⁺]_e, the responses were normalized to the EPSC₁. (D) Quantification of absolute values of 50 EPSCs elicited at 10 Hz recorded from neurons expressing either STX1A^WT, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆N2-28}. (E) Example traces (left) and quantification (right) of Ca²⁺-sensitivity recorded from neurons expressing STX1A^{LEOpen + ∆N2-19}, compared to that of neurons expressing STX1A^WT or STX1A^LEOpen. The responses were normalized to the response at [Ca²⁺]_e of 10 mM. The artifacts are blanked in the example traces. (F) Example traces (left) and quantifications (right) of STP measured at [Ca²⁺]_e of either 0.5 mM, 2 mM, or 10 mM by five stimulations at 40 Hz from neurons expressing either STX1A^WT, STX1A^{LEOpen + ∆N2-9}, or STX1A^{LEOpen + ∆N2-28}. The artifacts are blanked in the example traces. At every [Ca²⁺]_e, the responses were normalized to the EPSC₁. Data information: data points in all the graphs represent the mean ± SEM. Black and red annotations (stars and n.s.) on the graphs show the significance comparisons to STX1A^WT and STX1A^LEOpen rescue, respectively (nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). The numerical values are summarized in Figure 6—figure supplement 1—source data 1.

Figure 6—figure supplement 2.. Zoomed-in example traces of STP.

(A) Zoomed-in example traces of the first and last five EPSCs recorded by 50 stimulations at 10 Hz from STX1A^WT, STX1A^∆N2-9, STX1A^∆N2-19, or STX1A^∆N2-28 neurons. The traces show the absolute values. (B) Zoomed-in example traces of the first and last five EPSCs recorded by 50 stimulations at 10 Hz from STX1A^LEOpen, STX1A^{LEOpen + ∆N2-9}, STX1A^{LEOpen + ∆N2-19}, or STX1A^{LEOpen + ∆N2-28} neurons. The traces show the absolute values.

Figure 7.. Ca²⁺-influx is reduced in STX1-null and in STX1^LEOpen neurons.

(A, B) Example images and average of SynGCaMP6f fluorescence as (ΔF/F₀) in STX1-null neurons either not rescued or rescued with STX1A^WT, STX1A^∆N2-9, STX1A^∆N2-19, STX1A^LEOpen, or STX1A^{LEOpen + ∆N2-28}. The images were recorded at baseline, and at 1, 2, 5, 10, and 20 action potentials (APs). Scale bar: 10 µm (C–E) Maximum fluorescence changes (ΔF/F₀) in STX1-null, STX1A^∆N2-9, STX1A^∆N2-28, STX1A^LEOpen, or STX1A^{LEOpen + ∆N2-28} in comparison to that in STX1A^WT neurons recorded at 1, 2, 5, 10, and 20 APs. (F–I-J) Summary plots of STX1A expression level, neuronal viability, readily releasable pool (RRP) charge, number of docked synaptic vesicles (SVs), and maximum SynGCaMP6f ΔF/F₀ at 20 AP from STX1-null, STX1A^WT, TX1A^∆N2-28, STX1A^LEOpen, and STX1A^{LEOpen + ∆N2-28}. All the values were normalized to the one obtained from STX1A^WT neurons in each individual culture. Data information: data points in all graphs represent the mean ± SEM. Black annotations on the graphs show the significance comparisons to STX1A^WT (either unpaired t-test or Mann–Whitney test was applied in C based on the normality of the data; in D and E, nonparametric Kruskal–Wallis test followed by Dunn’s post hoc test was applied, *p≤0.05, ****p≤0.0001). The numerical values are summarized in Figure 7—source data 1.

Figure 7—figure supplement 1.. Reducing the expression level of STX1A^WT does not alter Ca²⁺-influx.

(A, B) Example images and average of SynGCaMP6f fluorescence as (ΔF/F₀) in STX1-null neurons either not rescued or rescued with 1× or 1/12× viral volume for STX1A^WT. The images were recorded at baseline, and at 1, 2, 5, 10, and 20 action potentials (APs). Scale bar:10 µm. (C) Maximum fluorescence changes (ΔF/F₀) in STX1A^WT 1× and 1/12× neurons recorded at 1, 2, 5, 10, and 20 APs. Data information: data points in all graphs represent the mean ± SEM. Black annotation on the graph shows the significance comparison (Mann–Whitney test was applied). The numerical values are summarized in Figure 7—figure supplement 1—source data 1.

Figure 8.. Speculative model of effects of N-peptide deletions and LE_Open mutation on vesicular release.

(A) Native state of STX1A. (B) N-peptide deletion of STX1A leads to a decrease in Ca²⁺-sensitivity of vesicular release and short-term depression (STD) upon 10 Hz stimulation potentially through increased distance of Ca²⁺-channel synaptic vesicle (SV) coupling. (C) LE_Open mutation on STX1A increases fusogenicity and Ca²⁺-sensitivity of SVs and thus leads to a high degree of STD. It also leads to reduced global Ca²⁺-influx. (D) SV fusion proceeds normal when LE_Open mutation is combined with N-peptide deletion. LE_Open mutation dictates SV fusogenicity and Ca²⁺-influx by increasing the former and decreasing the latter.