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Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells

Nature Neuroscience volume 5, pages 649–656 (2002)Cite this article

Abstract

Ca2+-triggered dense-core vesicle exocytosis in PC12 cells does not require vesicular synaptotagmins 1 and 2, but may use plasma membrane synaptotagmins 3 and 7 as Ca2+ sensors. In support of this hypothesis, C2 domains from the plasma membrane but not vesicular synaptotagmins inhibit PC12 cell exocytosis. Ca2+ induces binding of both plasma membrane and vesicular synaptotagmins to phospholipids and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors), although with distinct apparent Ca2+ affinities. Here we used gain-of-function C2-domain mutants of synaptotagmin 1 and loss-of-function C2-domain mutants of synaptotagmin 7 to examine how synaptotagmins function in dense-core vesicle exocytosis. Our data indicate that phospholipid- but not SNARE-binding by plasma membrane synaptotagmins is the primary determinant of Ca2+-triggered dense-core vesicle exocytosis. These results support a general lipid-based mechanism of action of synaptotagmins in exocytosis, with the specificity of various synaptotagmins for different types of fusion governed by their differential localizations and Ca2+ affinities.

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Figure 1: Sequence determinants of the Ca2+ affinities of C2 domains.
Figure 2: Phospholipid-specificity during Ca2+-dependent phospholipid binding of various synaptotagmin 1 and 7 C2A-domain mutants.
Figure 3: Apparent Ca2+ affinities of wild-type and mutant synaptotagmin 1 and 7 C2A domains measured with a resin-pulldown assay.
Figure 4: Ca2+-dependent binding of wild-type and mutant synaptotagmin 1 and 7 C2A domains to the SNARE proteins synaptobrevin/VAMP 2, syntaxin 1 and SNAP-25.
Figure 5: Comparative analysis the inhibition of Ca2+-triggered exocytosis in cracked PC12 cells by wild type and mutant C2A domains.
Figure 6: Ca2+-dependence of exocytosis in cracked PC12 cells in the presence of wild-type and mutant C2A domains from synaptotagmin 1 and 7.
Figure 7: Comparison of Ca2+-dependent phospholipid binding by the C2A domains of synaptotagmins 1 (Syt 1-C2A) and 7 (Syt 7-C2A) and the C2 domain of cytoplasmic phospholipase A2 (cPLA2-C2): effects of phospholipid composition.
Figure 8: Effects of the cPLA2 C2 domain on Ca2+-triggered exocytosis in cracked PC12 cells.

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Acknowledgements

We thank I. Leznicki, A. Roth and E. Borowicz for technical assistance, and S. Gerber for the phospholipase A2 C2-domain expression plasmid. This study was supported by grants from the NIH to J.R. (NS37200 and NS40944).

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  1. Department of Molecular Genetics and Howard Hughes Medical Institute, Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, 75390, Texas, USA

    Ok-Ho Shin & Thomas C. Südhof

  2. Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, 75390, Texas, USA

    Josep Rizo

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Correspondence to Thomas C. Südhof.

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Shin, OH., Rizo, J. & Südhof, T. Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells. Nat Neurosci 5, 649–656 (2002). https://doi.org/10.1038/nn869

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