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Direct interaction of SNARE complex binding protein synaphin/complexin with calcium sensor synaptotagmin 1

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Brain Cell Biology

Abstract

Although the binding of synaphin (also called complexin) to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is critical for synaptic vesicle exocytosis, the exact role of synaphin remains unclear. Here, we show that synaphin directly binds to synaptotagmin 1, a major Ca2+ sensor for fast neurotransmitter release, in a 1:1 stoichiometry. Mapping of the synaphin site involved in synaptotagmin 1 binding revealed that the C-terminal region is essential for the interaction between these two proteins. Binding was sensitive to ionic strength, suggesting the involvement of charged residues in the C-terminus region. Mutation of the seven consecutive glutamic acid residues (residues 108–114) at the C-terminal region of synaphin to alanines or glutamines resulted in a dramatic reduction in synaptotagmin 1 binding activity. Furthermore, a peptide from the C-terminus of synaphin (residues 91–124) blocked the binding of synaptotagmin 1 to synaphin, an effect that was abolished by mutating the consecutive glutamic acid residues to alanine. Immunoprecipitation experiments with brain membrane extracts showed the presence of a complex consisting of synaphin, synaptotagmin 1, and SNAREs. We propose that synaphin recruits synaptotagmin 1 to the SNARE-based fusion complex and synergistically functions with synaptotagmin 1 in mediating fast synaptic vesicle exocytosis.

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Acknowledgments

We thank M. Takahashi (Kitasato University, Sagamihara, Japan) for supplying the monoclonal antibodies to Syt1 and syntaxin 1, and M. Watanabe (Tokushima Bunri University) for mass spectrometric analysis of the bound proteins. We also thank Y. Li (Stanford University) for his critical reading of this manuscript. This work was supported in part by grants (to H. T., and T. A.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and from the Takeda Science Foundation.

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Kagawa, 769-2193, Japan

    Hiroshi Tokumaru & Chigusa Shimizu-Okabe

  2. Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, 951-8585, Japan

    Teruo Abe

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  1. Hiroshi Tokumaru
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  2. Chigusa Shimizu-Okabe
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  3. Teruo Abe
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Correspondence to Hiroshi Tokumaru.

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Tokumaru, H., Shimizu-Okabe, C. & Abe, T. Direct interaction of SNARE complex binding protein synaphin/complexin with calcium sensor synaptotagmin 1. Brain Cell Bio 36, 173–189 (2008). https://doi.org/10.1007/s11068-008-9032-9

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