Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo

Abstract

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca2+ sensor for fast, evoked neurotransmitter release1. Deleting the gene for synaptotagmin (sytnull) strongly suppresses synaptic transmission in every species examined2, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C2 domains, C2A and C2B. Five, highly conserved, acidic residues in both the C2A and C2B domains of synaptotagmin coordinate the binding of Ca2+ ions3,4,5, and biochemical studies have characterized several in vitro Ca2+-dependent interactions between synaptotagmin and other nerve terminal molecules6. But there has been no direct evidence that any of the Ca2+-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca2+-binding aspartate residues in the C2B domain (D416,418N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca2+ affinity of evoked transmitter release. These studies show that the Ca2+-binding motif of the C2B domain of synaptotagmin is essential for synaptic transmission.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Fast, Ca2+-evoked synaptic transmission is nearly abolished, and the apparent Ca2+ affinity of evoked transmitter release is decreased, in a C2B Ca2+-binding motif mutant.
Figure 2: Expression of B-D3,4N or B-D1,2N mutant synaptotagmin inhibits synaptic transmission in otherwise wild-type synapses.
Figure 3: Synaptotagmin expression in the nervous system of P[sytWT], P[sytB-D3,4N] and P[sytB-D1,2N] transgenic lines.
Figure 4: Whereas defects in recycling and Ca2+-dependent oligomerization cannot account for the severe decrease in synaptic transmission, decreased Ca2+-dependent phospholipid binding may account for this.

Similar content being viewed by others

References

  1. Augustine, G. J. How does calcium trigger neurotransmitter release? Curr. Opin. Neurobiol. 11, 320–326 (2001)

    Article  CAS  PubMed  Google Scholar 

  2. Lin, R. C. & Scheller, R. H. Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16, 19–49 (2000)

    Article  CAS  PubMed  Google Scholar 

  3. Shao, X., Davletov, B. A., Sutton, R. B., Sudhof, T. C. & Rizo, J. Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C. Science 273, 248–251 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Sutton, R. B., Ernst, J. A. & Brunger, A. T. Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III: Implications for Ca+ 2-independent SNARE complex interaction. J. Cell Biol. 147, 589–598 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fernandez, I. et al. Three-dimensional structure of the synaptotagmin 1 C2B-domain: Synaptotagmin 1 as a phospholipid binding machine. Neuron 32, 1057–1069 (2001)

    Article  CAS  PubMed  Google Scholar 

  6. Rizo, J. & Südhof, T. C2-domains, structure and function of a universal Ca2+-binding domain. J. Biol. Chem. 273, 15879–15882 (1998)

    Article  CAS  PubMed  Google Scholar 

  7. Bommert, K. et al. Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature 363, 163–165 (1993)

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Fukuda, M. et al. Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal. Proc. Natl Acad. Sci. USA 92, 10708–10712 (1995)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Desai, R. C. et al. The C2B domain of synaptotagmin is a Ca2+-sensing module essential for exocytosis. J. Cell Biol. 150, 1125–1136 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Littleton, J. T. et al. synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. J. Neurosci. 21, 1421–1433 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mackler, J. M. & Reist, N. E. Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions. J. Comp. Neurol. 436, 4–16 (2001)

    Article  CAS  PubMed  Google Scholar 

  12. Li, C. et al. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375, 594–599 (1995)

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Bai, J., Wang, P. & Chapman, E. R. C2A activates a cryptic Ca2+-triggered membrane penetration activity within the C2B domain of synaptotagmin I. Proc. Natl Acad. Sci. USA 99, 1665–1670 (2002)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Loewen, C. A., Mackler, J. M. & Reist, N. E. Drosophila synaptotagmin I null mutants survive to early adulthood. Genesis 31, 30–36 (2001)

    Article  CAS  PubMed  Google Scholar 

  15. Petersen, S. A., Fetter, R. D., Noordermeer, J. N., Goodman, C. S. & DiAntonio, A. Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron 19, 1237–1248 (1997)

    Article  CAS  PubMed  Google Scholar 

  16. Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J. & Wu, C.-F. Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J. Comp. Physiol. A 175, 179–191 (1994)

    Article  CAS  PubMed  Google Scholar 

  17. Zhang, J. Z., Davletov, B. A., Sudhof, T. C. & Anderson, R. G. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78, 751–760 (1994)

    Article  CAS  PubMed  Google Scholar 

  18. Jorgensen, E. M. et al. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature 378, 196–199 (1995)

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Reist, N. E. et al. Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila. J. Neurosci. 18, 7662–7673 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kee, Y. & Scheller, R. H. Localization of synaptotagmin-binding domains on syntaxin. J. Neurosci. 16, 1975–1981 (1996)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Earles, C. A., Bai, J., Wang, P. & Chapman, E. R. The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis. J. Cell Biol. 154, 1117–1123 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fernandez-Chacon, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Robinson, I. M., Ranjan, R. & Schwarz, T. L. Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain. Nature advance online publication, 7 July 2002 (doi:10.1038/nature00915)

  24. Garcia, R. A., Forde, C. E. & Godwin, H. A. Calcium triggers an intramolecular association of the C2 domains in synaptotagmin. Proc. Natl Acad. Sci. USA 97, 5883–5888 (2000)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. DiAntonio, A., Parfitt, K. D. & Schwarz, T. L. Synaptic transmission persists in synaptotagmin mutants of Drosophila. Cell 73, 1281–1290 (1993)

    Article  CAS  PubMed  Google Scholar 

  26. Del Castillo, J. & Katz, B. Quantal components of the end-plate potential. J. Physiol. 124, 560–573 (1954)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Davletov, B. A. & Sudhof, T. C. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding. J. Biol. Chem. 268, 26386–26390 (1993)

    CAS  PubMed  Google Scholar 

  28. Littleton, J. T., Bellen, H. J. & Perin, M. S. Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse. Development 118, 1077–1088 (1993)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank S. Royer, C. Williams and K. Mace for technical assistance, and R. Handa, K. Beam, J. Herbers, M. Tamkun and R. Aldrich for discussions about this manuscript. This work was supported by the National Science Foundation (N.E.R.), the Muscular Dystrophy Association (N.E.R.), the March of Dimes (N.E.R.) and an MRC Career Development Award (I.M.R.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to N. E. Reist.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mackler, J., Drummond, J., Loewen, C. et al. The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418, 340–344 (2002). https://doi.org/10.1038/nature00846

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature00846

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing