STX2 supports neuronal viability but does not rescue synchronous evoked release, the RRP or the clamping of spontaneous release.

A. STX1A and STX2 domain structure scheme and SNARE domain sequence alignment (68% homology). Layers are highlighted in gray.

B. Example images of high-densitiy cultured STX1-null hippocampal neurons rescued with GFP (STX1-null), STX1A or STX2 (from top to bottom) at DIV8, DIV15, DIV22 and DIV29 (from left to right). Immunofluorescent labelling of MAP2. Scale bar: 500μm.

C. Quantification of total number of neurons at DIV8 of each group.

D. Quantification of the percentage of the surviving neurons at DIV8, DIV15, DIV22 and DIV29 normalized to the number of neurons at DIV8 in the same group.

E. Example traces (left) and quantification of EPSC amplitude (right) from autaptic Stx1A-null hippocampal mouse neurons rescued with STX1A or STX2.

F. Quantification of the cumulative charge transfer of the EPSC from the onset of the response until 0.55s after.

G. Example traces of normalized EPSC to their peak amplitude (left) and quantification of the half width of the EPSC (right).

H. Example traces (left) and quantification of the response induced by a 5s 0.5M application of sucrose, which represents the readily releasable pool of vesicles (RRP).

I. Example traces (left) and quantification of the frequency of the miniature excitatory postsynaptic currents (mEPSC) (rights).

J. Quantification of the vesicle release probability (PVR) as the ratio of the EPSC charge over the RRP charge (PVR).

K. Quantification of the spontaneous vesicular release rate as the ratio between the of mEPSC frequency and number of vesicles in the RRP.

L. Example traces (left) and the quantification of a 40Hz paired-pulse ratio (PPR).

Data information: In (D) data points represent mean ± SEM. In (D, E-L) data is show in a whisker-box plot. Each data point represents single observations, middle line represents the median, boxes represent the distribution of the data and external data points represent outliers. In (C, D) significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.001, ***p≤0.001, ****p≤0.0001. In (E-L) Significances and P values of data were determined by non-parametric Mann-Whitney test and unpaired two-tailed t-test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 1 – Source Data 1.

The C-terminal half of the SNARE domain of STX1A has a regulatory effect on the RRP, spontaneous release and both, N- and C-terminus have a role in the regulation of efficacy of Ca2+-evoked release.

A. STX1A WT and chimera domain structure scheme, sequence alignment of STX1A and STX2 SNARE domain and percentage homology between both SNARE domains.

B. Example traces (left) and quantification of the EPSC amplitude (right) from autaptic STX1A-null hippocampal mouse neurons rescued with STX1A, STX1A-2(SNARE), STX1A-2(Nter) or STX1A-2(Cter).

C. Quantification of the cumulative charge transfer of the EPSC from the onset of the response until 0.55s after.

D. Example traces of normalized EPSC to their peak amplitude (left) and quantification of the half width of the EPSC (right).

E. Example traces (left) and quantification of the frequency of the miniature excitatory postsynaptic currents (mEPSC) (right).

F. Example traces (left) and quantification of the response induced by a 5s 0.5M application of sucrose, which represents the readily releasable pool of vesicles (RRP).

G. Quantification of the vesicle release probability (PVR) as the ratio of the EPSC charge over the RRP charge (PVR).

H. Quantification of the spontaneous vesicular release rate as the ratio between the of mEPSC frequency and number of vesicles in the RRP.

I. Example traces (left) and the quantification of a 40Hz paired-pulse ratio (PPR).

J. Quantification of STP measured by 50 stimulations at 10Hz.

Data information: In (B, D-I) data is show in a whisker-box plot. Each data point represents single observations, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. In (J) data represents the mean ± SEM. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 1 – Source Data 2.

The C-terminal half of the SNARE domain of STX1A has a regulatory effect on the spontaneous release and the RRP and the speed of Ca2+-evoked release depends on the integrity of the SNARE domain.

A. STX2 WT and chimera domain structure scheme, sequence alignment of STX2 and STX1A SNARE domain and percentage homology between both SNARE domains.

B. Example traces (left) and quantification of the EPSC amplitude (right) from autaptic STX1A-null hippocampal mouse neurons rescued with STX2, STX2-1A(SNARE), STX2-1A(Nter) or STX2-1A(Cter).

C. Quantification of the cumulative charge transfer of the EPSC from the onset of the response until 0.55s after.

D. Example traces of normalized EPSC to their peak amplitude (left) and quantification of the half width of the EPSC (right).

E. Example traces (left) and quantification of the frequency of the miniature excitatory postsynaptic currents (mEPSC) (right).

F. Example traces (left) and quantification of the response induced by a 5s 0.5mM application of sucrose, which represents the readily releasable pool of vesicles (RRP).

G. Quantification of the vesicle release probability (PVR) as the ratio of the EPSC charge over the RRP charge (PVR).

H. Quantification of the spontaneous vesicular release rate as the ratio between the of mEPSC frequency and number of vesicles in the RRP.

I. Example traces (left) and the quantification of a 40Hz paired-pulse ratio (PPR).

J. Quantification of STP measured by 50 stimulations at 10Hz.

Data Information: In (E-L) data is show in a whisker-box plot. Each data point represents single observations, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 3 – Source Data 1.

Quantification of STX1A and Munc18-1 levels at the synapse.

A. Example images of Stx1-null neurons plated in high-density cultures and rescued with STX1A, STX1A-2(SNARE), STX1A-2(Nter) or STX1A-2(Cter) or GFP (STX1-null) as negative control. Neurons were fixed between DIV14-16. Cultures were stained with fluorophore-labeled antibodies that recognize VGlut1 (red in merge), STX1A (green in merge) and Munc18-1(blue in merge), from left to right. Scale bar: 10μm.

B. Quantification of the immunofluorescent intensity of STX1A normalized to the intensity of the same VGlut1-labeled ROIs. Values where normalized to values in STX1A rescue group.

C. Quantification of the immunofluorescent intensity of Munc18-1 normalized to the intensity of the same VGlut1-labeled ROIs. Values where normalized to values in STX1 rescue group.

D. SDS-PAGE of the electrophoretic analysis of neuronal lysates obtained from each experimental group. Proteins were detected using antibodies that recognize β-Tubuline III as loading control, STX1A and Munc18-1.

E. Correlation between Munc18-1 values and EPSC amplitude of Stx1-null neurons expressing STX1A, STX2, STX2-1A(SNARE), STX2-1A(Nter) or STX2-1A(Cter).

F. Correlation between Munc18-1 values and RRP.

G. Correlation between Munc18-1 values and PVR.

H. Correlation between Munc18-1 values and spontaneous vesicular release rate

Data information: In (B, C) data is show in a whisker-box plot. Each data point represents single ROIs, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. In (E-H) each data point is the correlation of the mean ±SEM. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 4 – Source Data 1.

Quantification of STX2 and Munc18-1 levels at the synapse.

A. Example images of Stx1-null neurons plated in high-density cultures and rescued with STX1A, STX2, STX2-1A(SNARE), STX2-1A(Nter), STX2-1A(Cter) or GFP (STX1-null) as negative control. Between DIV14-16 neurons were fixed. (A) Example images of neurons stained with fluorophore-labeled antibodies that recognize VGlut1 (red in merge) and STX2 (green in merge), from left to right. Scale bar: 10μm.

B. Quantification of the immunofluorescent intensity of STX2 normalized to the intensity of the same VGlut1-labeled ROIs. Values where normalized to values in STX2 rescue group.

C. SDS-PAGE of the electrophoretic analysis of neuronal lysates obtained from each experimental group. Proteins were detected using antibodies that recognize β-Tubuline III as loading control and STX2.

D. SDS-PAGE of the electrophoretic analysis of neuronal lysates obtained from each experimental group Proteins were detected using antibodies that recognize β-Tubuline III and Munc18-1.

E. Example images of neurons stained with fluorophore-labeled antibodies that recognize VGlut1 (red in merge) and Munc18-1(blue in merge), from left to right. Scale bar: 10μm.

F. Quantification of the immunofluorescent intensity of Munc18-1 normalized to the intensity of the same VGlut1-labeled ROIs. Values where normalized to values in STX2 rescue group.

G. Correlation between Munc18-1 values and EPSC amplitude of STX1-null neurons expressing STX1A, STX2, STX2-1A(SNARE), STX2-1A(Nter) or STX2-1A(Cter).

H. Correlation between Munc18-1 values and RRP.

I. Correlation between Munc18-1 values and PVR.

J. Correlation between Munc18-1 values and spontaneous vesicular release rate.

Data information: In (B, F) data is show in a whisker-box plot. Each data point represents single ROIs, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. In (G-J) each data point is the correlation of the mean ±SEM. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 5 – Source Data 1.

The charge of the outer-surface residues in the C-terminal half of the SNARE domain is important for clamping spontaneous release and D231,R232 are important in the stabilization of the pool and the efficiency of Ca2+-evoked release.

A. Sequence of the C-terminal half of STX1A and STX2 and single- and double-point mutations in the sequence of STX1A WT.

B. Example traces (left) and quantification of the EPSC amplitude (right) from autaptic STX1A-null hippocampal mouse neurons rescued with STX1A, STX1AD231N, STX1AR232N, STX1AY235R, STX1AE238V, STX1AV248K, STX1AS249E STX1AD231N,R232N and STX1AV248K,S249E.

C. Example traces (left) and quantification of the response induced by a 5s 0.5M application of sucrose, which represents the readily releasable pool of vesicles (RRP).

D. Quantification of the vesicle release probability (PVR) as the ratio of the EPSC charge over the RRP charge (PVR).

E. Example traces (left) and quantification of the frequency of the miniature excitatory postsynaptic currents (mEPSC) (right).

F. Quantification of the spontaneous vesicular release rate as the ratio between the of mEPSC frequency and number of vesicles in the RRP.

G. Quantification of the spontaneous vesicular release rate as the ratio between the of mEPSC frequency and number of vesicles in the RRP but STX1A-2(SNARE) and STX1A-2(Cter) chimeras were removed.

H. Correlation between the RRP and the spontaneous vesicular release rate. Both values are normalized to STX1A WT.

I. Correlation between the PVR and the spontaneous vesicular release rate. Values are normalized to STX1A WT.

Data information: (B-H) All electrophysiological recording were done on autaptic neurons. Between 30-35 neurons per group from 3 independent cultures where recorded. Values from STX1A-2(SNARE) and STX1A-2(Cter) groups were taken from the experiments done in Figure 2 and normalized to their own STX1A WT control and used here for visual comparison. Data points represent the mean ±SEM. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 6 – Source Data 1.

Speculative model based on most important changes in electrophysiological properties found in STX1A-chimeras or STX2-chimeras compared to their WT isoforms.

A. Energy landscape for priming and fusion of synaptic vesicles that have SNARE complexes formed with STX1A WT (black line), STX1A-2(SNARE or Cter) (red dotted line), STX2 WT (blue line); STX2-1A(Cter) (light green dotted line).

B. Summarized conclusions of our results based on the speculative model in (A).

C. Changes in the equilibrium between the unprimed/primed state (reflecting the stability of the primed state) and the rate of fusion (energy landscape for fusion adapted from “Sørensen, 2009”).

STX2 is expressed in STX1-null hippocampal neurons.

A. SDS-PAGE electrophoresis of lysates from STX1-null neurons infected with STX1A, STX2 or GFP (STX1-null) and STX3A as negative controls. Proteins were detected using antibodies that recognize β-Tubuline III as loading control STX1A, STX2 and STX3A.

B. Example images of mass-culture hippocampal neurons stained with fluorophore-labeled antibodies that recognize VGlut1 (red in merge) and STX2 (green in merge) from left to right. Scale bar: 10μm.

C. Quantification of the immunofluorescent intensity of STX2 normalized to the intensity of the same VGlut1-labeled ROIs.

Data information: In (C) data is show in a whisker-box plot. Each data point represents single ROIs, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 1 – Figure Supplement 1 – Source Data 1.

Quantification of kinetic parameters of the EPSC and the mEPSC of autaptic STX1A-null hippocampal mouse neurons rescued with STX1A, STX1A-2(SNARE), STX1A-2(Nter) or STX1A-2(Cter).

A. Quantification of the rise time (20-80%) of the EPSC neurons

B. Quantification of the decay time (80-20%) of the EPSC.

C. Quantification of the mEPSC charge.

D. Quantification of the mEPSC amplitude.

E. Quantification of the rise time (20-80%) of the mEPSC.

F. Quantification of the half width of the mEPSC.

G. Quantification of the decay time of the mEPSC.

H. All electrophysiological recording were done on autaptic neurons.

Data information: Data in (A-G) is show in a whisker-box plot. Each data point represents single observations, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test or by Ordinary one-way ANOVA; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 2 – figure Supplement 1 – Source Data 1

Quantification of kinetic parameters of the EPSC and the mEPSC of autaptic STX1A-null hippocampal mouse neurons rescued with STX2, STX2-1A(SNARE), STX2-1A(Nter) or STX2-1A(Cter).

A. Quantification of the rise time (20-80%) of the EPSC.

B. Quantification of the decay time (80-20%) of the EPSC.

C. Quantification of the mEPSC charge.

D. Quantification of the mEPSC amplitude.

E. Quantification of the rise time (20-80%) of the mEPSC.

F. Quantification of the half width of the mEPSC.

G. Quantification of the decay time of the mEPSC.

Data information: Data in (A-G) is show in a whisker-box plot. Each data point represents single observations, middle line represents the median, boxes represent the distribution of the data, where the majority of the data points lie and external data points represent outliers. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test or by Ordinary one-way ANOVA; **p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 3 – Figure Supplement 1 – Source Data 1.

Quantification of STX2 levels at the synapse.

A. Example images of Stx1-null neurons plated in high-density cultures and rescued with STX1A, STX2, STX2-1A(SNARE), STX2-1A(Nter), STX2-1A(Cter) or GFP (STX1-null) as negative control. Between DIV14-16 neurons were fixed. Neurons were stained with fluorophore-labeled antibodies that recognize VGlut1 (red in merge), STX1A (green in merge) and Munc18-1 (blue in merge) from left to right. Scale bar: 10μm.

B. Quantification of the immunofluorescent intensity of STX1A normalized to the intensity of the same VGlut1-labeled ROIs. Values where normalized to STX1A WT values.

C. Quantification of the immunofluorescent intensity of Munc18-1 normalized to the intensity of the same VGlut1-labeled ROIs. Values where normalized to STX1A WT values.

D. SDS-PAGE of the electrophoretic analysis of neuronal lysates obtained from each experimental group. Proteins were detected using antibodies that recognize β-Tubuline III as loading control and STX1A and Munc18-1.

Data information: In (B, C) data points represent the mean ±SEM. 7-9 images per group per culture were obtained and 3-5 ROI per image where analyzed. 3 replicates per group. Significances and P values of data were determined by non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. All data values are summarized in Figure 6 – Figure Supplement 1 – Source Data 1.