DOI: 10.5061/dryad.np5hqc05k
title: Data from: Synaptotagmin-1 serves as a primary Zn<sup>2+</sup> sensor to mediate spontaneous neurotransmitter release under pathological conditions

description: This dataset provides the source data for the paper titled
 "Synaptotagmin-1 serves as a primary Zn²⁺ sensor to mediate
 spontaneous neurotransmitter release under pathological conditions."
 Data includes electrophysiological patch-clamp recordings of miniature
 excitatory postsynaptic currents (mEPSCs), spontaneous EPSCs (sEPSCs), and
 evoked EPSCs, along with single-vesicle and ensemble lipid-mixing assays
 (in vitro reconstitution system) measuring fusion probability (%),
 cumulative probability, and lipid-mixing efficiency (FRET). The dataset
 also covers zinc-protein or membrane-binding assays evaluating binding
 affinity and membrane-binding capacity, as well as vesicle docking imaging
 via TIRF microscopy, which quantifies docking-related fluorescence
 intensity and the number of docked vesicles. Data collection and analysis
 were conducted collaboratively across multiple institutions.
 Electrophysiological patch-clamp recordings and vesicle docking imaging
 were performed at the Key Laboratory of Xi'an Jiaotong University,
 while the remaining data were collected at the relevant experimental
 facilities of Sichuan University.
description: # Data from: Synaptotagmin-1 serves as a primary Zn^2+^ sensor to mediate
 spontaneous neurotransmitter release under pathological conditions Dataset
 DOI: [10.5061/dryad.np5hqc05k](https://doi.org/10.5061/dryad.np5hqc05k) ##
 Description of the data and file structure ### Files and variables #
 **Description of the data and file structure** This dataset
 (Source_data.zip) comprises four integrated experimental modules:
 electrophysiology, in vitro vesicle fusion reconstitution, zinc ion
 binding and membrane interaction analysis, and total internal reflection
 fluorescence (TIRF) microscopy imaging. Electrophysiological recordings
 from hippocampal neurons and slices—including miniature excitatory
 postsynaptic currents (mEPSC), spontaneous EPSC (sEPSC), and evoked
 EPSC—as well as vesicle docking imaging via TIRF microscopy, were
 conducted at the Key Laboratory of Xi'an Jiaotong University. In
 vitro single-vesicle and ensemble lipid-mixing assays, along with
 zinc-protein binding and membrane co-sedimentation experiments, were
 performed at relevant experimental facilities of Sichuan University. All
 raw data and statistical results are systematically organized by figure
 number in corresponding worksheets within a single Excel file (Source
 data.xlsx), ensuring full correspondence and traceability between data and
 figures. Analysis was carried out using GraphPad Prism 8.0, ImageJ, and
 OriginPro 2024. Detailed experimental procedures and analytical workflows
 are provided in the "Methods" section and supplementary
 materials of the associated paper. ## Code/software **File: Source
 data.xlsx** **Figure1c, d, f, h and i** **Description:**  This file
 provides electrophysiology data from hippocampal neurons measuring
 spontaneous(mEPSC) and evoked (EPSC) release frequencies and amplitudes
 under varying Zn²⁺ concentrations. **Variables** **Figure 1c and d :** *
 Zn²⁺ concentration (µM) – Treatment with different extracellular zinc ion
 concentrations (unit: micromolar). * mEPSC frequency (Hz) – Frequency of
 miniature excitatory postsynaptic currents in wild-type Syt1 neurons
 (unit: hertz). - mEPSC amplitude (pA) – Amplitude of miniature excitatory
 postsynaptic currents in wild-type Syt1 neurons (unit: picoampere).
 **Figure 1f :** * Zn²⁺ concentration (µM) – Treatment with different
 extracellular zinc ion concentrations (unit: micromolar). * Evoked EPSC
 amplitude (pA) – Amplitude of evoked excitatory postsynaptic currents in
 wild-type Syt1 neurons (unit: picoampere). **Figure 1h and i :** * Zn²⁺
 concentration (µM) – Treatment with different extracellular zinc ion
 concentrations (unit: micromolar). * mEPSC frequency (Hz) – Frequency of
 miniature excitatory postsynaptic currents in Syt1 conditional knockout
 neurons (unit: hertz). * mEPSC amplitude (pA) – Amplitude of miniature
 excitatory postsynaptic currents in Syt1 conditional knockout neurons
 (unit: picoampere). **Figure 2b,c, d, e, f and g** **Description:** This
 file provides the probability distribution and probability of vesicle
 fusion mediated by different conditions. **Variables** **Figure 2b and c
 :** * Time (s) – Recording time point (unit: seconds). * 500 μM
 Ion-dependent fusion probability – probability distribution of fusion
 events per second, recorded over 1 minute in the presence of
 ions(Sr²⁺、Mg²⁺、Ca²⁺、Zn²⁺) (unitless, Probability scale: Normalized
 cumulative probability (0-1 )). * Cumulative probability – Cumulative
 probability of ions(Sr²⁺、Mg²⁺、Ca²⁺、Zn²⁺)-dependent vesicle fusion events
 (unitless). * Zn²⁺ – Reaction progress treatmented by Zinc ions. * Ca²⁺ –
 Reaction progress treatmented by Calcium ions . * Mg²⁺ – Reaction progress
 treatmented by Magnesium ions. - Sr²⁺ – Reaction progress treatmented by
 Strontium ions. **Figure 2d and e :** * Time (s) – Recording time point
 (unit: seconds). * Zn²⁺ concentrations (µM)– 0、10、20、50、100 and 500 µM
 zinc ion (unit: micromolar). * Zn²⁺ -dependent fusion probability –
 probability distribution of fusion events per second, recorded over 1
 minute in the presence of ions (unitless, Probability scale: Normalized
 cumulative probability (0-1 )). - Cumulative probability – Cumulative
 probability of different Zn²⁺ concentrations(0、10、20、50、100 and 500
 µM)-dependent vesicle fusion events (unitless). **Figure 2f and g :** *
 Time (s) – Recording time point (unit: seconds). * 500 µM Zn²⁺-dependent
 fusion probability – probability distribution of fusion events per second,
 recorded over 1 minute in the presence of Syt1 and its
 mutants(Syt1_WT、Syt1_C2A**B**、Syt1_4A、Syt1_4W) (unitless, Probability
 scale: Normalized cumulative probability (0-1 )). * Syt1_WT – wild-type
 synaptotagmin-1  reconstituted into vesicles reconstituted into vesicles.
 * Syt1_C2A*B* -C2 domain calcium-binding mutant  reconstituted into
 vesicles reconstituted into vesicles. * Syt1_4A - Syt1 membrane-insertion
 mutant  reconstituted into vesicles reconstituted into vesicles. *
  Syt1_4W - Syt1 membrane-insertion mutant  reconstituted into vesicles
 reconstituted into vesicles. * Cumulative probability – 500 µM
 Zn²⁺-dependent fusion probability of Syt1 and its
 mutants(Syt1_WT、Syt1_C2A*B*、Syt1_4A、Syt1_4W) (unitless). **Figure3b,c, f
 and g** **Description:**  This file provides fluorescence spectroscopy
 data from FluoZin-1 assays for Zn²⁺ binding to the Syt1 C2AB and variants.
 **Variables** **Figure 3b, c and f :** * None – Buffer-only control (no
 protein). * C2AB – Full cytoplasmic domain fragment of synaptotagmin-1. *
 C2AB(Zn²⁺) – C2AB fragment pre-incubated with zinc ions. * C2A – Isolated
 C2A domain fragment of synaptotagmin-1. * C2B – Isolated C2B domain
 fragment of synaptotagmin-1. * C2A**B** – C2AB mutant with disrupted
 calcium-binding sites. * C2AB_3M – C2AB mutant with disrupted zinc-binding
 residues (D261A/E386A/H389A). * Em. Wavelength (nm) – Emission peak
 wavelength in fluorescence measurements (unit: nanometers). * Intensity
 (Counts/s/µA) – Fluorescence signal intensity measured at the
 corresponding peak wavelength (unit: arbitrary units(normalized
 fluorescence intensity)). * SE – Standard Error of the Mean for
 Intensity(unit: Same as the Intensity (unitless)). **Figure 3g:** *
 Concentration (µM) – Protein concentration in titration experiments (
 unit: micromolar). * Binding Affinity (Kd) – Dissociation constant from
 titration curves ( unit: nanomolar or micromolar). * C2AB: Full
 cytoplasmic domain fragment of synaptotagmin-1. * C2AB_3M – C2AB mutant
 with disrupted zinc-binding residues (D261A/E386A/H389A). * Intensity
 (a.u.) – Fluorescence signal intensity measured at the corresponding peak
 wavelength (unit: arbitrary units(normalized fluorescence intensity)). *
 SE – Standard Error of the Mean for Intensity(unit: Same as the Intensity
 (unitless)). **Figure 4a, b, d, e, g and h** **Description:** This file
 provides probability of vesicle fusion mediated by Syt1_WT or Syt1_3M and
 electrophysiology data from Syt1 cKO hippocampal neurons rescued by
 Syt1_WT or Syt1_3M measuring spontaneous(mEPSC). **Variables** **Figure 4a
 and b:** * Time (s) – Recording time point (unit: seconds). * 500 nM or
 500 µM Zn²⁺-dependent fusion probability – probability distribution of
 fusion events per second, recorded over 1 minuten the presence of Syt1_WT
 or Syt1_3M  (unitless, Probability scale: Normalized cumulative
 probability (0-1 )). * Syt1_WT – wild-type Syt1 protein reconstituted into
 vesicles reconstituted into vesicles. * Syt1_3M – Syt1 zinc-binding mutant
 protein  reconstituted into vesicles reconstituted into vesicles. *
 Cumulative probability – Cumulative probability of 500 nM or 500 µM
 Zn²⁺-dependent vesicle fusion events (unitless) **Figure 4d, e, g and h**
 * Zn²⁺ concentration (µM) – Treatment with different extracellular zinc
 ion concentrations (unit: micromolar). * cKO + Syt1_WT – Syt1 cKO
 hippocampal neurons rescued by Syt1_WT. * cKO + Syt1_3M –Syt1 cKO
 hippocampal neurons rescued by Syt1_3M. * mEPSC frequency (Hz) – Frequency
 of miniature excitatory postsynaptic currents in wild-type Syt1 neurons
 (unit: hertz). * mEPSC amplitude (pA) – Amplitude of miniature excitatory
 postsynaptic currents in wildtype Syt1 neurons (unit: picoampere).
 **Figure 5d, e and f** **Description:**  This file provides gel images and
 precipitation fluorescence spectra related to the study of the binding
 ability of Zn²⁺ to membranes containing different compositions of
 phosphatidylserine (PS) and phosphatidylinositol (PIP2). **Variables** *
 (0, 5, 10 and 15) PS concentration (%) – Phosphatidylserine content in
 lipid vesicles (unit: percentage). * 1 PIP₂ concentration (%) –
 Phosphatidylinositol 4,5-bisphosphate content in lipid vesicles (unit:
 percentage). * 15 PS + 1 PIP₂ concentration (%) – Phosphatidylserine and
 Phosphatidylinositol 4,5-bisphosphate content in lipid vesicles (unit:
 percentage). * Zn²⁺ concentration (µM) – Concentration of zinc ions in the
 experimental solution (unit: micromolar). * C2AB – Cytosolic tandem
 C2-domains of synaptotagmin-1. * C2AB_3M – Zinc-binding deficient triple
 mutant (D261A/E386A/H389A). * S – Supernatant in fraction. * P – Pellet in
 fraction. * Intensity (Counts/s/µA) – Fluorescence signal intensity
 measured at the corresponding peak wavelength (unit: arbitrary
 units(normalized fluorescence intensity)). * Normalized P/S – Normalized
 ratio of protein bound to the pellet fraction versus remaining in the
 supernatant fraction. * KDa– Kilo-Dalton, measuring protein mass. **Figure
 6c, d and f** **Description:**  This file provides on the quantity of
 vesicle docking promoted by Zn²⁺ in vitro and the changes in cellular
 imaging fluorescence intensity. **Variables** **Figure 6c and d:** * WT –
 wild-type hippocampal neurons. * Syt1cKO – Syt1 cKO hippocampal neurons. *
 Zn²⁺ concentration (µM) – Treatment with different extracellular zinc ion
 concentrations (unit: micromolar). * Time (s) – Elapsed time from the
 start of the recording.(unit: seconds (s)). * Intensity –  The
 fluorescence intensity trace for NH₄Cl perfusion pulse. * I₀ –  The
 fluorescence intensity trace for the first NH₄Cl perfusion pulse (before
 Zn²^+^ application). The peak value in this phase is defined as I₀(unit:
 Arbitrary Fluorescence Units (a.u.)). * I~Zn~²^+^ –  The fluorescence
 intensity trace from neurons treated with 200 µM Zn²^⁺^. The trace shows
 the response to NH₄Cl after zinc application. The peak in this phase is
 defined as I_Zn²^⁺^(unit: Arbitrary Fluorescence Units (a.u.)). *
 Intensity ratio (I₀ /I~Zn~²^+^ ) – dividing the maximal fluorescence
 intensity obtained during the first ammonium chloride (NH₄Cl) perfusion
 (I₀) by the maximal fluorescence intensity obtained during the second
 NH₄Cl perfusion performed after the application of Zn²⁺ (I_Zn²⁺). **Figure
 6f:** * Syt1_WT – Wild-type full-length Syt1 reconstituted into vesicles.
 * Syt1_QM –synaptotagmin-1 primary interface with the SNARE complex region
 mutant reconstituted into vesicles. * Syt1_QQQ  – synaptotagmin-1
 polybasic region mutant mutant reconstituted into vesicles. * Syt1_3M 
 –Syt1 zinc-binding mutant reconstituted into vesicles reconstituted into
 vesicles. * Zn²⁺ concentration (µM) – Treatment with 0 or 500 µM zinc ion
 concentrations (unit: micromolar). * Counts – the number of SV vesicles
 bound to PM vesicles. **Supplementary Data 1b and c**
 **Description:**  This file provides electrophysiology data from
 hippocampal neurons measuring spontaneous(sEPSC) release frequencies and
 amplitudes under varying Zn²⁺ concentrations. **Variables** * Zn²⁺
 concentration (µM) – Treatment with different extracellular zinc ion
 concentrations (unit: micromolar). * sEPSC frequency (Hz) – Frequency of
 spontaneous excitatory postsynaptic Current in wild-type Syt1 neurons
 (unit: hertz). * sEPSC amplitude (pA) – Amplitude of spontaneous
 excitatory postsynaptic currents in wild-type Syt1 neurons (unit:
 picoampere). **Supplementary Data 2b and c** **Description:**  This file
 provides electrophysiology data from hippocampal slices measuring
 spontaneous(mEPSC) release frequencies and amplitudes under varying Zn²⁺
 concentrations. **Variables** * Zn²⁺ concentration (µM) – Treatment with
 different extracellular zinc ion concentrations (unit: micromolar). *
 mEPSC frequency (Hz) – Frequency of miniature excitatory postsynaptic
 currents in hippocampal slices (unit: hertz). * mEPSC amplitude(pA) –
 Amplitude of miniature excitatory postsynaptic currents in hippocampal
 slices (unit: picoampere). **Supplementary Data 3b, c and e**
 **Description:**  This file provides electrophysiology data from
 hippocampal neurons measuring spontaneous(mEPSC) and evoked (EPSC) release
 frequencies and amplitudes in the absence of Ca^2+^ . **Variables**
 **Supplementary Data 3b and c :** * Zn²⁺ concentration (µM) – Treatment
 with different extracellular zinc ion concentrations (unit: micromolar). *
 Ca²⁺ concentration (µM) – Treatment with different extracellular calcium
 ion concentrations (unit: micromolar). * mEPSC frequency (Hz) – Frequency
 of miniature excitatory postsynaptic currents in wild-type Syt1 neurons
 (unit: hertz). * mEPSC amplitude (pA) – Amplitude of miniature excitatory
 postsynaptic currents in wild-type Syt1 neurons (unit: picoampere).
 **Supplementary Data 3e :** * Zn²⁺ concentration (µM) – Treatment with
 different extracellular zinc ion concentrations (unit: micromolar). * Ca²⁺
 concentration (µM) – Treatment with different extracellular calcium ion
 concentrations (unit: micromolar). * Evoked EPSC amplitude (pA) –
 Amplitude of evoked excitatory postsynaptic currents in wild-type Syt1
 neurons (unit: picoampere). **Supplementary Data 4b and c**
 **Description:**  This file provides electrophysiology data from
 hippocampal slices measuring spontaneous(sEPSC) release frequencies and
 amplitudes under varying Zn²⁺ concentrations. **Variables** * Zn²⁺
 concentration (µM) – Treatment with different extracellular zinc ion
 concentrations (unit: micromolar). * mEPSC frequency (Hz) – Frequency of
 spontaneous spontaneous postsynaptic currents in Syt1 conditional knockout
 neurons (unit: hertz). * mEPSC amplitude (pA) – Amplitude of spontaneous
 spontaneous postsynaptic currents in Syt1 conditional knockout neurons
 (unit: picoampere). **Supplementary Data 5a and b** **Description:**  This
 file provides the distribution and efficiency changes of Zn²⁺-dependent
 vesicle fusion with varying protein types, using single-vesicle fusion
 assay. **Variables** * Time (s) – Recording time point (unit: seconds). *
 Zn²⁺-dependent fusion probability – probability distribution of fusion
 events per second, recorded over 1 minute in the presence of ions
 (unitless, Probability scale: Normalized cumulative probability (0-1 )). *
 Cumulative probability – Cumulative probability of Zn²⁺-dependent vesicle
 fusion events by different protein types (unitless). * Syt1_WT – Wild-type
 Synaptotagmin-1 reconstituted into vesicles. This is the positive control
 with all necessary components. * C2AB – Only the cytosolic tandem C2
 domains of Syt1. * No Syt1 – Vesicles reconstituted without
 Synaptotagmin-1. * No VAMP2 – Vesicles lacking Vesicle-Associated Membrane
 Protein 2 (Synaptobrevin). - No SNAP-25 – Vesicles lacking
 Synaptosomal-Associated Protein 25 kDa. **Supplementary Data 6b and c**
 **Description:**  This file provides the maximum fusion
 probability(FRET-based) and efficiency changes of vesicle fusion dependent
 on different divalent ion,using lipid mixing assay **Variables** * Time
 (s) – Recording time point (unit: seconds) * Zn²⁺ – Reaction progress
 treated by Zinc ions * Ca²⁺ – Reaction progress treated by Calcium ions  *
 Mg²⁺ – Reaction progress treated by Magnesium ions * Sr²⁺ – Reaction
 progress treated by Strontium ions * Maximum fusion probability – Measured
 at the corresponding ions(unit: Unitless probability (range 0 to 1, where
 0 = no fusion, 1 = all vesicles fuse).) * SE – Standard Error of the Mean
 for the Maximum Fusion Probability(unit: Same as the probability
 (unitless)) **Supplementary Data 7a and b** **Description:**  This file
 provides the maximum fusion probability(FRET-based) and efficiency changes
 of vesicle fusion dependent on Zn^2+^ and Ca^2+^,using lipid mixing assay.
 **Variables** * Time (s) – Recording time point (unit: seconds). * None
 –Reaction progress treated  by no ions. * Zn²⁺ – Reaction progress
 treatmented by Zinc ions. * Ca²⁺ – Reaction progress treatmented by
 Calcium ions. * Zn^2+^ and Ca^2+^ – Sequence where Zinc is added first,
 followed by Calcium. * Ca^2+^ and Zn^2+^ –Sequence where Calcium is added
 first, followed by Zinc. * Maximum fusion probability – Measured at the
 corresponding Zn^2+^ and Ca^2+^(unit: Unitless probability (range 0 to 1,
 where 0 = no fusion, 1 = all vesicles fuse)) SE: Standard Error of the
 Mean for the Maximum Fusion Probability(unit: Same as the probability
 (unitless)). **Supplementary Data 8a and b** **Description:**  This file
 provides the distribution and efficiency changes of Ca²⁺-dependent vesicle
 fusion with varying protein types, using single-vesicle fusion assay.
 **Variables** * Time (s) – Recording time point (unit: seconds). *
 Ca²⁺-dependent fusion – probability distribution of fusion events per
 second, recorded over 1 minute in the presence of ions (unitless,
 Probability scale: Normalized cumulative probability (0-1 )). * Cumulative
 probability – Cumulative probability of Ca²⁺-dependent vesicle fusion
 events by different protein types (unitless). * Syt1_WT – Wild-type
 Synaptotagmin-1 reconstituted into vesicles. This is the positive control
 with all necessary components. * Syt1_C2A**B**
 – synaptotagmin-1 calcium-binding sites mutant reconstituted into
 vesicles. * Syt1_4A – synaptotagmin-1 -membrane-insertion
 mutant reconstituted into vesicles. * Syt1_4W – synaptotagmin-1
 -membrane-insertion mutant reconstituted into vesicles. **Supplementary
 Data 10** **Description:**  This file provides CD spectrum data for C2AB
 and its variant proteins. **Variables** * C2AB – Full cytoplasmic domain
 fragment of synaptotagmin-1. * C2A – Isolated C2A domain fragment. * C2B –
 Isolated C2B domain fragment. * C2A**B** – C2AB mutant with disrupted
 calcium-binding sites. * C2AB_3M – C2AB mutant with disrupted zinc-binding
 residues (D261A/E386A/H389A). * wavelength [nm] – The wavelength of
 light(unit - Nanometers) **Supplementary Data 11a, b, c and d**
 **Description:**  This file provides the maximum fusion
 probability(FRET-based) and efficiency changes of vesicle fusion dependent
 on different Zn^2+^ concentration,using lipid mixing assay. **Variables**
 * Time (s) – Recording time point (unit: seconds). -
 Zn^2+^ concentration(nM) – Treatment with different zinc ion
 concentrations (unit: nanomolar). - Syt1_WT – Wild-type Synaptotagmin-1
 reconstituted into vesicles. - Syt1_3M – Syt1 zinc-binding mutant
 reconstituted into vesicles. - Syt1_WT(2 mM Mg^2+^) – Wild-type
 Synaptotagmin-1 reconstituted into vesicles with 2 mM Mg^2+^ in solution.
 - Syt1_3M(2 mM Mg^2+^) – Syt1 zinc-binding mutant reconstituted into
 vesicles with 2 mM Mg^2+^ in solution. - Maximum fusion probability –
 Measured at the corresponding different zinc ion concentrations (unit:
 Unitless probability (range 0 to 1, where 0 = no fusion, 1 = all vesicles
 fuse)). * SE: Standard Error of the Mean for the Maximum Fusion
 Probability(unit: Same as the probability (unitless)). **Supplementary
 Data 12a and b** **Description:**  This file provides the maximum fusion
 probability(FRET-based) and efficiency changes of vesicle fusion dependent
 on Ca^2+^ concentration,using lipid mixing assay. **Variables** * Time (s)
 – Recording time point (unit: seconds). * Syt1_WT – Wild-type
 Synaptotagmin-1 reconstituted into vesicles. * Syt1_3M – Syt1 zinc-binding
 mutant reconstituted into vesicles. * Syt1_WT(Ca^2+^) – Wild-type
 Synaptotagmin-1 reconstituted into vesicles treatmented with Ca^2+^. *
 Syt1_3M(Ca^2+^) – Syt1 zinc-binding mutant reconstituted into
 vesicles treatmented with Ca^2+^. * Maximum fusion probability – Measured
 at the corresponding Ca^2+^(unit: Unitless probability (range 0 to 1,
 where 0 = no fusion, 1 = all vesicles fuse)). * SE: Standard Error of the
 Mean for the Maximum Fusion Probability(**unit:** Same as the probability
 (unitless)). **Supplementary Data 13b** **Description:**  This file
 provides electrophysiology data from Syt1 cKO hippocampal neurons rescued
 by Syt1_3M measuring evoked(EPSC) release frequencies and amplitudes under
 varying Zn²⁺ concentrations. **Variables** * Zn²⁺ concentration (µM) –
 Treatment with different extracellular zinc ion concentrations (unit:
 micromolar). * Evoked EPSC amplitude (pA) – Amplitude of evoked excitatory
 postsynaptic currents in Syt1 cKO hippocampal neurons rescued by Syt1_3M
 (unit: picoampere). **Supplementary Data 14b, c, e and f**
 **Description:**  This file provides electrophysiology data from Syt1 cKO
 hippocampal neurons rescued by Syt1_WT or Syt1_3M measuring
 spontaneous(sEPSC) release frequencies and amplitudes under varying Zn²⁺
 concentrations. **Variables** * Zn²⁺ concentration (µM) – Treatment with
 different extracellular zinc ion concentrations (unit: micromolar). * cKO
 + Syt1_WT –Syt1 cKO hippocampal neurons rescued by Syt1_WT. * sEPSC
 frequency (Hz) – Frequency of spontaneous excitatory postsynaptic currents
 in Syt1 cKO hippocampal neurons rescued by Syt1_WT neurons(unit: hertz). *
 sEPSC amplitude (pA) – Amplitude of spontaneous excitatory postsynaptic
 currents in Syt1 cKO hippocampal neurons rescued by Syt1_WT neurons(unit:
 picoampere). * cKO + Syt1_3M –Syt1 cKO hippocampal neurons rescued by
 Syt1_3M. * sEPSC frequency (Hz)– Frequency of spontaneous excitatory
 postsynaptic currents in Syt1 cKO hippocampal neurons rescued by Syt1_3M
 neurons(unit: hertz). * sEPSC amplitude (pA) – Amplitude of spontaneous
 excitatory postsynaptic currents in Syt1 cKO hippocampal neurons rescued
 by Syt1_3M neurons (unit: picoampere). **Supplementary Data 15a, b and c**
 **Description:**  This file provides gel images and precipitation
 fluorescence spectra related to the study of the binding ability of Ca²⁺
 to membranes containing compositions of phosphatidylserine (PS) and
 phosphatidylinositol (PIP2). **Variables** * Ca²⁺ concentration (µM) –
 Concentration of Calcium ions in the experimental solution (unit:
 micromolar). * C2AB – Cytosolic tandem C2-domains of synaptotagmin-1. *
 C2A*B*– C2AB mutant with disrupted calcium-binding sites. * C2AB_QQQ –
 C2AB polybasic region mutant. * S – Supernatant in fraction. * P – Pellet
 in fraction. * KDa– Kilo-Dalton, measuring protein mass. * 15% PS + 1%
 PIP2 – The lipid composition of the vesicles included 15%
 phosphatidylserine (PS) and 1% phosphatidylinositol 4,5-bisphosphate
 (PIP₂). * Intensity (Counts/s/µA) – Fluorescence signal intensity measured
 at the corresponding peak wavelength (unit: arbitrary units(normalized
 fluorescence intensity)). * wavelength [nm] – The wavelength of light(unit
 - Nanometers). Normalized P/S – Normalized ratio of protein bound to the
 pellet fraction versus remaining in the supernatant fraction.
 **Supplementary Data 16a, b and c** **Description:**  This file provides
 gel images and precipitation fluorescence spectra related to the study of
 the binding ability of Zn²⁺ to membranes containing compositions of
 phosphatidylserine (PS) and phosphatidylinositol (PIP2). **Variables** *
 Zn²⁺ concentration (µM) – Concentration of zinc ions in the experimental
 solution (unit: micromolar). * C2AB – Cytosolic tandem C2-domains of
 synaptotagmin-1. * C2A*B*– C2AB mutant with disrupted calcium-binding
 sites. * C2AB_QQQ – C2AB polybasic region mutant. * S – Supernatant in
 fraction. * P – Pellet in fraction. * KDa– Kilo-Dalton, measuring protein
 mass. * 15% PS + 1% PIP2 – The lipid composition of the vesicles included
 15% phosphatidylserine (PS) and 1% phosphatidylinositol 4,5-bisphosphate
 (PIP₂). * Intensity (Counts/s/µA) – Fluorescence signal intensity measured
 at the corresponding peak wavelength (unit: arbitrary units(normalized
 fluorescence intensity)). * wavelength [nm] – The wavelength of light(unit
 - Nanometers). - Normalized P/S – Normalized ratio of protein bound to the
 pellet fraction versus remaining in the supernatant fraction.
 **Supplementary Data 17a and b** **Description:**  This file provides the
 fluorescence response curve and quantitated values for Zn²⁺ influx in
 hippocampal neurons. **Variables** * Time (s) – Recording time point
 (unit: seconds). * Intensity – Fluorescence intensity change of the Zn²⁺
 probe in the selected region(Supplementary Data 17a). - Intensity –
 Fluorescence values at 0 seconds and 120 seconds(Supplementary Data 17b).
 **Supplementary Data 18a and b** **Description:**  This file provides the
 distribution and efficiency changes of Zn²⁺-dependent vesicle fusion with
 varying protein types, using single-vesicle fusion assay. **Variables** *
 Time (s) – Recording time point (unit: seconds) * 500 μM Zn²⁺-dependent
 fusion probability – probability distribution of fusion events per second,
 recorded over 1 minute in the presence of 500 μM Zn²⁺ (unitless,
 Probability scale: Normalized cumulative probability (0-1 )). * Cumulative
 probability – Cumulative probability of Zn²⁺-dependent vesicle fusion
 events by different protein types (unitless). * Syt1_WT – Wild-type
 Synaptotagmin-1 reconstituted into vesicles. This is the positive control
 with all necessary components. * Syt1_QM – synaptotagmin-1 primary
 interface with the SNARE complex region mutant reconstituted into
 vesicles. * Syt1_QQQ  – synaptotagmin-1 polybasic region mutant
 mutant reconstituted into vesicles. * Syt1_3M  –Syt1 zinc-binding mutant
 reconstituted into vesicles reconstituted into vesicles. ## Access
 information Other publicly accessible locations of the data: * The complex
 of Syt1/Syt3 and Zn^2+^ data generated in this study have been deposited
 in the ModelArchive database under accession codes ma-sle84, ma-36fbl.
 Data was derived from the following sources: * The crystal structure of
 the C2AB fragment of Syt3 used in this study were obtained from the
 Protein Data Bank (PDB) with accession code 3HN8.