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Synaptotagmin I functions as a calcium regulator of release probability

Nature volume 410, pages 41–49 (2001)Cite this article

Abstract

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors—which are the gatekeepers to neurotransmission—remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

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Figure 1: Architecture of the Ca2+-binding sites at the top of the C2A domain of synaptotagmin I.
Figure 2: Ca2+ titrations of wild-type and mutant C2A domains monitored by 1H–15N HSQC spectra.
Figure 3: Ca2+ dependence of phospholipid binding to wild-type and mutant C2A domains.
Figure 4: Generation of mice with mutant C2A domains.
Figure 5: Effect of the R233Q mutation on Ca2+-dependent phosphopholipid and syntaxin binding by native synaptotagmin.
Figure 6: Effect of the R233Q and K236Q mutations on synaptic responses in cultured hippocampal neurons.
Figure 7: Synaptic release probability in hippocampal neurons from R233Q knockin and wild-type knockin mice.
Figure 8: Pool sizes and Ca2+ responsiveness of synaptic vesicles in wild-type, R233Q and K236Q mutant synapses.

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Acknowledgements

We thank I. Herfort, I. Leznicki and A. Roth for technical assistance; H. Riedesel, J. Krause, S. Röcklin and the ARC in Dallas for help with mouse husbandry; and E. Neher, G. Alvarez de Toledo, J. López-Barneo and R. Jahn for advice. This study was supported by grants from the NIH to J.R., a grant from the Deutsche Forschungsgemeinschaft to C.R., Heisenberg Fellowships from the Deutsche Forschungsgemeinschaft to N.B. and C.R., a grant from the Perot Family Foundation to T.C.S., and postdoctoral fellowships from the Spanish Ministry of Education and the Fulbright Commission to R.F.C., and from the Deutsche Forschungsgemeinschaft to S.H.G.

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Author notes

  1. Rafael Fernández-Chacón, Andreas Königstorfer and Charles F. Stevens: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Molecular Genetics, Center for Basic Neuroscience, and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, 75390-9111, Texas, USA

    Rafael Fernández-Chacón, Stefan H. Gerber, Maria F. Matos & Thomas C. Südhof

  2. Max-Planck-Institut für experimentelle Medizin,

    Andreas Königstorfer & Nils Brose

  3. Max-Planck-Institut für biophysikalische Chemie, Göttingen, 37070, Germany

    Rafael Fernández-Chacón & Christian Rosenmund

  4. Departments of Biochemistry and Pharmacology, The University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Jesús García & Josep Rizo

  5. The Salk Institute, and Howard Hughes Medical Institute, La Jolla, 92037, California, USA

    Charles F. Stevens

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  1. Rafael Fernández-Chacón
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Correspondence to Thomas C. Südhof.

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Fernández-Chacón, R., Königstorfer, A., Gerber, S. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001). https://doi.org/10.1038/35065004

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