Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Nov 7;92(23):10703–10707. doi: 10.1073/pnas.92.23.10703

Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

K Mikoshiba 1, M Fukuda 1, J E Moreira 1, F M Lewis 1, M Sugimori 1, M Niinobe 1, R Llinás 1
PMCID: PMC40680  PMID: 7479868

Abstract

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Full text

PDF
10703

Images in this article

10704Fig. 1
on p.10704
10705Fig. 2
on p.10705
10705Fig. 3
on p.10705
10706Fig. 4
on p.10706
10707Fig. 5
on p.10707

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bommert K., Charlton M. P., DeBello W. M., Chin G. J., Betz H., Augustine G. J. Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature. 1993 May 13;363(6425):163–165. doi: 10.1038/363163a0. [DOI] [PubMed] [Google Scholar]
  2. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  3. Davletov B. A., Südhof T. C. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding. J Biol Chem. 1993 Dec 15;268(35):26386–26390. [PubMed] [Google Scholar]
  4. Fukuda M., Aruga J., Niinobe M., Aimoto S., Mikoshiba K. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J Biol Chem. 1994 Nov 18;269(46):29206–29211. [PubMed] [Google Scholar]
  5. Fukuda M., Moreira J. E., Lewis F. M., Sugimori M., Niinobe M., Mikoshiba K., Llinás R. 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 U S A. 1995 Nov 7;92(23):10708–10712. doi: 10.1073/pnas.92.23.10708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Li C., Ullrich B., Zhang J. Z., Anderson R. G., Brose N., Südhof T. C. Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins. Nature. 1995 Jun 15;375(6532):594–599. doi: 10.1038/375594a0. [DOI] [PubMed] [Google Scholar]
  7. Llinás R., Steinberg I. Z., Walton K. Presynaptic calcium currents in squid giant synapse. Biophys J. 1981 Mar;33(3):289–321. doi: 10.1016/S0006-3495(81)84898-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Llinás R., Sugimori M., Lang E. J., Morita M., Fukuda M., Niinobe M., Mikoshiba K. The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12990–12993. doi: 10.1073/pnas.91.26.12990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Llinás R., Sugimori M., Silver R. B. Microdomains of high calcium concentration in a presynaptic terminal. Science. 1992 May 1;256(5057):677–679. doi: 10.1126/science.1350109. [DOI] [PubMed] [Google Scholar]
  10. Llinás R., Sugimori M., Silver R. B. Time resolved calcium microdomains and synaptic transmission. J Physiol Paris. 1995;89(2):77–81. doi: 10.1016/0928-4257(96)80554-7. [DOI] [PubMed] [Google Scholar]
  11. Niinobe M., Yamaguchi Y., Fukuda M., Mikoshiba K. Synaptotagmin is an inositol polyphosphate binding protein: isolation and characterization as an Ins 1,3,4,5-P4 binding protein. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1036–1042. doi: 10.1006/bbrc.1994.2770. [DOI] [PubMed] [Google Scholar]
  12. Perin M. S., Fried V. A., Mignery G. A., Jahn R., Südhof T. C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 1990 May 17;345(6272):260–263. doi: 10.1038/345260a0. [DOI] [PubMed] [Google Scholar]
  13. Scheller R. H. Membrane trafficking in the presynaptic nerve terminal. Neuron. 1995 May;14(5):893–897. doi: 10.1016/0896-6273(95)90328-3. [DOI] [PubMed] [Google Scholar]
  14. Schweizer F. E., Betz H., Augustine G. J. From vesicle docking to endocytosis: intermediate reactions of exocytosis. Neuron. 1995 Apr;14(4):689–696. doi: 10.1016/0896-6273(95)90213-9. [DOI] [PubMed] [Google Scholar]
  15. Südhof T. C. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. doi: 10.1038/375645a0. [DOI] [PubMed] [Google Scholar]
  16. Zhang J. Z., Davletov B. A., Südhof T. C., Anderson R. G. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell. 1994 Sep 9;78(5):751–760. doi: 10.1016/s0092-8674(94)90442-1. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES