The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release rely on the integrity of the C-terminal half of the SNARE domain of syntaxin-1A
- Department of Neurophysiology, Charité-Universitätsmedizin Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Germany
- NeuroCure Excellence Cluster, Germany
Figures
Figure 1 with 1 supplement
STX2 supports neuronal viability but does not rescue synchronous evoked release, the readily releasable pool (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-density cultured STX1-null hippocampal …
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Figure 1—source data 1
Quantification of the neuronal density at different time intervals and quantification of neurotransmitter release parameters of STX1-null neurons transduced with STX1A and STX2.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
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 …
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Figure 1—figure supplement 1—source data 1
Quantification of STX1A and STX2 levels in STX1-null neurons.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Whole SDS-PAGE image represented in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
The C-terminal half of the SNARE domain of STX1A has a regulatory effect on the readily releasable pool (RRP), spontaneous release, and both, and 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 …
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Figure 2—source data 1
Quantification of neurotransmitter release parameters of STX1-null neurons transduced with STX1A, STX1A-2(SNARE), STX1A-2(Nter) and STX1A-2(Cter).
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Quantification of kinetic parameters of the excitatory postsynaptic current (EPSC) and the miniature EPSC (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 …
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Figure 2—figure supplement 1—source data 1
Quantification of neurotransmitter release parameters of STX1-null neurons transduced with STX1A, STX1A-2(SNARE), STX1A-2(Nter) and STX1A-2(Cter).
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
The C-terminal half of the SNARE domain of STX1A has a regulatory effect on the spontaneous release and the readily releasable pool (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 …
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Figure 3—source data 1
Quantification of neurotransmitter release parameters of STX1-null neurons transduced with STX2, STX2-1A(SNARE), STX2-1A(Nter) and STX2-1A(Cter).
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Quantification of kinetic parameters of the excitatory postsynaptic current (EPSC) and the miniature EPSC (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 …
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Figure 3—figure supplement 1—source data 1
Quantification of neurotransmitter release parameters of STX1-null neurons transduced with STX2, STX2-1A(SNARE), STX2-1A(Nter) and STX2-1A(Cter).
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig3-figsupp1-data1-v1.xlsx
Figure 4
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 …
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Figure 4—source data 1
Quantification of STX1A and Munc18 levels in STX1-null neurons transduced with STX1A, STX1A-2(SNARE), STX1A-2(Nter) and STX1A-2(Cter).
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig4-data1-v1.xlsx
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Figure 4—source data 2
Whole SDS-PAGE image represented in Figure 4.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig4-data2-v1.zip
Figure 5
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. Neurons …
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Figure 5—source data 1
Quantification of STX2 and Munc18 levels in STX1-null neurons transduced with STX2, STX2-1A(SNARE), STX2-1A(Nter) and STX2-1A(Cter).
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig5-data1-v1.xlsx
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Figure 5—source data 2
Whole SDS-PAGE image represented in Figure 5.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig5-data2-v1.zip
Figure 6 with 1 supplement
The charge in 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 excitatory postsynaptic …
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Figure 6—source data 1
Quantification of neurotransmitter release parameters of STX1-null neurons transduced with STX1AD231N, STX1AR232N, STX1AY235R, STX1AE238V, STX1AV248K, STX1AS249E, STX1AD231N,R232N, and STX1AV248K,S249E.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig6-data1-v1.xlsx
Figure 6—figure supplement 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. Neurons …
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Figure 6—figure supplement 1—source data 1
Quantification of STX1A and Munc18-1 levels in STX1-null neurons transduced with STX1AD231N, STX1AR232N, STX1AY235R, STX1AE238V, STX1AV248K, STX1AS249E, STX1AD231N,R232N, and STX1AV248K,S249E.
- https://cdn.elifesciences.org/articles/90775/elife-90775-fig6-figsupp1-data1-v1.xlsx
Figure 7
Speculative model based on the most important changes in electrophysiological properties found in STX1A-chimeras or STX2-chimeras compared to their WT isoforms.
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), and …
