Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1

Rafael Fernández-Chacón, Ok Ho Shin, Andreas Königstorfer, Maria F. Matos, Alexander C. Meyer, Jesus Garcia, Stefan H. Gerber, Jose Rizo-Rey, Thomas C. Südhof, Christian Rosenmund

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Abstract

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+ dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.

Original languageEnglish (US)
Pages (from-to)8438-8446
Number of pages9
JournalJournal of Neuroscience
Volume22
Issue number19
StatePublished - Oct 1 2002

Fingerprint

Synaptotagmin I
Phospholipids
Mutation
Synaptic Vesicles
Exocytosis
Recombinant Proteins
Synaptic Transmission
Morbidity
Phenotype
Neurons
Mortality
C2 Domains

Keywords

  • C2 domain
  • Ca-binding site
  • Exocytosis
  • Neurotransmitter release
  • Synaptic plasticity
  • Synaptotagmin

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Fernández-Chacón, R., Shin, O. H., Königstorfer, A., Matos, M. F., Meyer, A. C., Garcia, J., ... Rosenmund, C. (2002). Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1. Journal of Neuroscience, 22(19), 8438-8446.

Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1. / Fernández-Chacón, Rafael; Shin, Ok Ho; Königstorfer, Andreas; Matos, Maria F.; Meyer, Alexander C.; Garcia, Jesus; Gerber, Stefan H.; Rizo-Rey, Jose; Südhof, Thomas C.; Rosenmund, Christian.

In: Journal of Neuroscience, Vol. 22, No. 19, 01.10.2002, p. 8438-8446.

Research output: Contribution to journalArticle

Fernández-Chacón, R, Shin, OH, Königstorfer, A, Matos, MF, Meyer, AC, Garcia, J, Gerber, SH, Rizo-Rey, J, Südhof, TC & Rosenmund, C 2002, 'Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1', Journal of Neuroscience, vol. 22, no. 19, pp. 8438-8446.
Fernández-Chacón R, Shin OH, Königstorfer A, Matos MF, Meyer AC, Garcia J et al. Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1. Journal of Neuroscience. 2002 Oct 1;22(19):8438-8446.
Fernández-Chacón, Rafael ; Shin, Ok Ho ; Königstorfer, Andreas ; Matos, Maria F. ; Meyer, Alexander C. ; Garcia, Jesus ; Gerber, Stefan H. ; Rizo-Rey, Jose ; Südhof, Thomas C. ; Rosenmund, Christian. / Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1. In: Journal of Neuroscience. 2002 ; Vol. 22, No. 19. pp. 8438-8446.
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abstract = "Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+ dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.",
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AU - Garcia, Jesus

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