Controlling synaptotagmin activity by electrostatic screening

Article metrics


Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca2+ sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+, but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca2+-dependent exocytosis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: SNARE-dependent fusion of CGs with LUVs.
Figure 2: Role of polyphosphates in Ca2+-dependent vesicle fusion as measured by a lipid-mixing assay.
Figure 3: Roles of synaptotagmin-1 and PI(4,5)P2 in Ca2+-dependent enhancement of vesicle fusion.
Figure 4: Effect of ATP on association of synaptotagmin-1 to the vesicle membrane.
Figure 5: Effect of acidic phospholipid concentration in the liposome membrane on Ca2+-dependent binding of synaptotagmin-1.
Figure 6: Effect of polyphosphates on Ca2+-dependent binding of synaptotagmins to membranes containing acidic phospholipids.


  1. 1

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

  2. 2

    De Camilli, P. & Jahn, R. Pathways to regulated exocytosis in neurons. Annu. Rev. Physiol. 52, 625–645 (1990).

  3. 3

    Park, Y. & Kim, K.T. Short-term plasticity of small synaptic vesicle (SSV) and large large dense-core vesicle (LDCV) exocytosis. Cell. Signal. 21, 1465–1470 (2009).

  4. 4

    Neher, E. Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20, 389–399 (1998).

  5. 5

    Neher, E. A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse. Pflugers Arch. 453, 261–268 (2006).

  6. 6

    Jahn, R. & Scheller, R.H. SNAREs—engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

  7. 7

    Martens, S. & McMahon, H.T. Mechanisms of membrane fusion: disparate players and common principles. Nat. Rev. Mol. Cell Biol. 9, 543–556 (2008).

  8. 8

    Martin, T.F. The molecular machinery for fast and slow neurosecretion. Curr. Opin. Neurobiol. 4, 626–632 (1994).

  9. 9

    Rizo, J. & Rosenmund, C. Synaptic vesicle fusion. Nat. Struct. Mol. Biol. 15, 665–674 (2008).

  10. 10

    Malsam, J., Kreye, S. & Sollner, T.H. Membrane fusion: SNAREs and regulation. Cell. Mol. Life Sci. 65, 2814–2832 (2008).

  11. 11

    Südhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

  12. 12

    Stein, A., Weber, G., Wahl, M.C. & Jahn, R. Helical extension of the neuronal SNARE complex into the membrane. Nature 460, 525–528 (2009).

  13. 13

    Wiederhold, K. & Fasshauer, D. Is assembly of the SNARE complex enough to fuel membrane fusion? J. Biol. Chem. 284, 13143–13152 (2009).

  14. 14

    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).

  15. 15

    Chapman, E.R. How does synaptotagmin trigger neurotransmitter release? Annu. Rev. Biochem. 77, 615–641 (2008).

  16. 16

    Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. & Jahn, R. Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat. Struct. Mol. Biol. 14, 904–911 (2007).

  17. 17

    Vennekate, W. et al. Cis- and trans-membrane interactions of synaptotagmin-1. Proc. Natl. Acad. Sci. USA 109, 11037–11042 (2012).

  18. 18

    Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

  19. 19

    Holt, M., Riedel, D., Stein, A., Schuette, C. & Jahn, R. Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+. Curr. Biol. 18, 715–722 (2008).

  20. 20

    Mahal, L.K., Sequeira, S.M., Gureasko, J.M. & Sollner, T.H. Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin 1. J. Cell Biol. 158, 273–282 (2002).

  21. 21

    Eaton, B.A., Haugwitz, M., Lau, D. & Moore, H.P. Biogenesis of regulated exocytotic carriers in neuroendocrine cells. J. Neurosci. 20, 7334–7344 (2000).

  22. 22

    Grabner, C.P., Price, S.D., Lysakowski, A. & Fox, A.P. Mouse chromaffin cells have two populations of dense-core vesicles. J. Neurophysiol. 94, 2093–2104 (2005).

  23. 23

    Plattner, H., Artalejo, A.R. & Neher, E. Ultrastructural organization of bovine chromaffin cell cortex—analysis by cryofixation and morphometry of aspects pertinent to exocytosis. J. Cell Biol. 139, 1709–1717 (1997).

  24. 24

    Kim, T., Gondre-Lewis, M.C., Arnaoutova, I. & Loh, Y.P. Dense-core secretory granule biogenesis. Physiology (Bethesda) 21, 124–133 (2006).

  25. 25

    Meldolesi, J., Chieregatti, E. & Luisa Malosio, M. Requirements for the identification of dense-core granules. Trends Cell Biol. 14, 13–19 (2004).

  26. 26

    Pobbati, A.V., Stein, A. & Fasshauer, D. N- to C-terminal SNARE complex assembly promotes rapid membrane fusion. Science 313, 673–676 (2006).

  27. 27

    Struck, D.K., Hoekstra, D. & Pagano, R.E. Use of resonance energy transfer to monitor membrane fusion. Biochemistry 20, 4093–4099 (1981).

  28. 28

    Chernomordik, L.V. et al. Lysolipids reversibly inhibit Ca2+-, GTP- and pH-dependent fusion of biological membranes. FEBS Lett. 318, 71–76 (1993).

  29. 29

    Lee, H.K. et al. Dynamic Ca2+-dependent stimulation of vesicle fusion by membrane-anchored synaptotagmin-1. Science 328, 760–763 (2010).

  30. 30

    Davletov, B.A. & Sudhof, T.C. A single C2 domain from synaptotagmin-1 is sufficient for high-affinity Ca2+-phospholipid binding. J. Biol. Chem. 268, 26386–26390 (1993).

  31. 31

    Zhang, X., Rizo, J. & Sudhof, T.C. Mechanism of phospholipid binding by the C2A-domain of synaptotagmin-1. Biochemistry 37, 12395–12403 (1998).

  32. 32

    Chapman, E.R. & Jahn, R. Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain. J. Biol. Chem. 269, 5735–5741 (1994).

  33. 33

    Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).

  34. 34

    Milosevic, I. et al. Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells. J. Neurosci. 25, 2557–2565 (2005).

  35. 35

    Radhakrishnan, A., Stein, A., Jahn, R. & Fasshauer, D. The Ca2+ affinity of synaptotagmin-1 is markedly increased by a specific interaction of its C2B domain with phosphatidylinositol-4,5-bisphosphate. J. Biol. Chem. 284, 25749–25760 (2009).

  36. 36

    van den Bogaart, G. et al. Membrane-protein sequestering by ionic protein-lipid interactions. Nature 479, 552–555 (2011).

  37. 37

    Botelho, R.J., Scott, C.C. & Grinstein, S. Phosphoinositide involvement in phagocytosis and phagosome maturation. Curr. Top. Microbiol. Immunol. 282, 1–30 (2004).

  38. 38

    Gillooly, D.J. et al. Localization of phosphatidylinositol-3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588 (2000).

  39. 39

    Lai, Y. & Shin, Y.K. The importance of an asymmetric distribution of acidic lipids for synaptotagmin-1 function as a Ca2+ sensor. Biochem. J. 443, 223–229 (2012).

  40. 40

    Bhalla, A., Chicka, M.C., Tucker, W.C. & Chapman, E.R. Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. 13, 323–330 (2006).

  41. 41

    Kyoung, M. et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl. Acad. Sci. USA 108, E304–E313 (2011).

  42. 42

    Wilson, J.E. & Chin, A. Chelation of divalent cations by ATP, studied by titration calorimetry. Anal. Biochem. 193, 16–19 (1991).

  43. 43

    Kuo, W., Herrick, D.Z., Ellena, J.F. & Cafiso, D.S. The calcium-dependent and calcium-independent membrane binding of synaptotagmin-1: two modes of C2B binding. J. Mol. Biol. 387, 284–294 (2009).

  44. 44

    Vrljic, M. et al. Post-translational modifications and lipid-binding profile of insect cell–expressed full-length mammalian synaptotagmin-1. Biochemistry 50, 9998–10012 (2011).

  45. 45

    Li, L. et al. Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin-1. J. Biol. Chem. 281, 15845–15852 (2006).

  46. 46

    Baker, P.F. & Knight, D.E. Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature 276, 620–622 (1978).

  47. 47

    Barszczewski, M. et al. A novel site of action for α-SNAP in the SNARE conformational cycle controlling membrane fusion. Mol. Biol. Cell 19, 776–784 (2008).

  48. 48

    van den Bogaart, G. et al. Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation. Nat. Struct. Mol. Biol. 18, 805–812 (2011).

  49. 49

    Smith, A.D. & Winkler, H. A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem. J. 103, 480–482 (1967).

  50. 50

    Li, Y. et al. A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain. Biochemistry 33, 7014–7020 (1994).

  51. 51

    Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

  52. 52

    Cypionka, A. et al. Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE proteins using FCS. Proc. Natl. Acad. Sci. USA 106, 18575–18580 (2009).

  53. 53

    Kendall, D.A. & MacDonald, R.C. Characterization of a fluorescence assay to monitor changes in the aqueous volume of lipid vesicles. Anal. Biochem. 134, 26–33 (1983).

Download references


We are indebted to G. Mieskes (Department of Neurobiology, Max Planck Institute for Biophysical Chemistry) for the arrangement of adrenal glands and logistical assistance. This work was supported by grants from the Alexander von Humboldt Foundation (to Y.P.) and the US National Institutes of Health (2 P01 GM072694-06A1 to R.J.).

Author information

J.M.H. assisted in the generation of SNARE-containing large unilamellar liposomes and performed the light-scattering experiments. G.v.d.B. provided labeled proteins and assisted in the fluorescence anisotropy experiments. S.A. and M.H. provided purified synaptic vesicles. D.R. performed EM. Y.P. and R.J. designed the study and wrote the paper. Experiments were conducted mainly by Y.P. All authors discussed the results and commented on the manuscript.

Correspondence to Reinhard Jahn.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 608 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Park, Y., Hernandez, J., van den Bogaart, G. et al. Controlling synaptotagmin activity by electrostatic screening. Nat Struct Mol Biol 19, 991–997 (2012) doi:10.1038/nsmb.2375

Download citation

Further reading