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Research Articles, Cellular/Molecular

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release

Zhenyong Wu, Lu Ma, Nicholas A. Courtney, Jie Zhu, Ane Landajuela, Yongli Zhang, Edwin R. Chapman and Erdem Karatekin
Journal of Neuroscience 27 July 2022, 42 (30) 5816-5829; DOI: https://doi.org/10.1523/JNEUROSCI.1385-21.2022
Zhenyong Wu
1Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06520
2Nanobiology Institute, Yale University, West Haven, Connecticut 06516
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Lu Ma
2Nanobiology Institute, Yale University, West Haven, Connecticut 06516
3Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06520
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Nicholas A. Courtney
4Howard Hughes Medical Institute and Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53705
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Jie Zhu
1Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06520
2Nanobiology Institute, Yale University, West Haven, Connecticut 06516
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Ane Landajuela
1Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06520
2Nanobiology Institute, Yale University, West Haven, Connecticut 06516
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Yongli Zhang
3Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06520
5Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
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Edwin R. Chapman
4Howard Hughes Medical Institute and Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53705
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Erdem Karatekin
1Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06520
2Nanobiology Institute, Yale University, West Haven, Connecticut 06516
5Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520
6Saints-Pères Paris Institute for the Neurosciences, Université de Paris, Centre National de la Recherche Scientifique UMR 8003, 75270 Paris, France
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Abstract

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain–membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain–membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

  • calcium-triggered exocytosis
  • exocytosis
  • membrane–protein interactions
  • neurotransmitter release
  • synaptotagmin

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The Journal of Neuroscience: 42 (30)
Journal of Neuroscience
Vol. 42, Issue 30
27 Jul 2022
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Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release
Zhenyong Wu, Lu Ma, Nicholas A. Courtney, Jie Zhu, Ane Landajuela, Yongli Zhang, Edwin R. Chapman, Erdem Karatekin
Journal of Neuroscience 27 July 2022, 42 (30) 5816-5829; DOI: 10.1523/JNEUROSCI.1385-21.2022

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Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release
Zhenyong Wu, Lu Ma, Nicholas A. Courtney, Jie Zhu, Ane Landajuela, Yongli Zhang, Edwin R. Chapman, Erdem Karatekin
Journal of Neuroscience 27 July 2022, 42 (30) 5816-5829; DOI: 10.1523/JNEUROSCI.1385-21.2022
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Keywords

  • calcium-triggered exocytosis
  • exocytosis
  • membrane–protein interactions
  • neurotransmitter release
  • synaptotagmin

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