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.