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
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
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
Similar content being viewed by others
References
Augustine, G. J. How does calcium trigger neurotransmitter release? Curr. Opin. Neurobiol. 11, 320–326 (2001)
Lin, R. C. & Scheller, R. H. Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16, 19–49 (2000)
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)
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)
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)
Rizo, J. & Südhof, T. C2-domains, structure and function of a universal Ca2+-binding domain. J. Biol. Chem. 273, 15879–15882 (1998)
Bommert, K. et al. Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature 363, 163–165 (1993)
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)
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)
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)
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)
Li, C. et al. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375, 594–599 (1995)
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)
Loewen, C. A., Mackler, J. M. & Reist, N. E. Drosophila synaptotagmin I null mutants survive to early adulthood. Genesis 31, 30–36 (2001)
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)
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)
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)
Jorgensen, E. M. et al. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature 378, 196–199 (1995)
Reist, N. E. et al. Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila. J. Neurosci. 18, 7662–7673 (1998)
Kee, Y. & Scheller, R. H. Localization of synaptotagmin-binding domains on syntaxin. J. Neurosci. 16, 1975–1981 (1996)
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)
Fernandez-Chacon, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001)
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)
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)
DiAntonio, A., Parfitt, K. D. & Schwarz, T. L. Synaptic transmission persists in synaptotagmin mutants of Drosophila. Cell 73, 1281–1290 (1993)
Del Castillo, J. & Katz, B. Quantal components of the end-plate potential. J. Physiol. 124, 560–573 (1954)
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)
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)
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
Corresponding author
Ethics declarations
Competing interests
The authors declare that they have no competing financial interests.
Supplementary information
Rights 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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature00846
This article is cited by
-
The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses
Communications Biology (2023)
-
Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration
Nature Communications (2023)
-
Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission
Scientific Reports (2021)
-
Function of Drosophila Synaptotagmins in membrane trafficking at synapses
Cellular and Molecular Life Sciences (2021)
-
Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain
Scientific Reports (2020)
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.