Reconstitution of Depolarization and Ca2+-Evoked Secretion in Xenopus Oocytes Monitored by Membrane Capacitance

  • Roy Cohen
  • Bernhard M. Schmitt
  • Daphne Atlas
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 440)

Summary

The identity of the proteins that constitute the “minimal molecular machinery” required for depolarization-evoked neurotransmitter release at synapses is still not fully disclosed. Using capacitance monitoring combined with heterologous protein expression in Xenopus oocytes, we were able to reconstitute a fast (<.5 s) secretion that was triggered directly by membrane depolarization. The functional assembly of voltage-gated Ca2+ channel (Cav1.2 or Cav2.2) coexpressed with syntaxin 1A, synaptosome-associated protein of 25kDa (SNAP-25), and synaptotagmin led to the reconstitution of depolarization-evoked secretion. Botulinum C1, botulinum A, and tetanus toxin were used to establish that this minimal set of proteins, named the excitosome complex, was necessary and sufficient for reconstituting depolarization-induced exocytosis. Similar to synaptic transmission, the capacitance changes were sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channels (Lc or N type; ionotropic glutamate GLUR3), and depended nonlinearly on extracellular divalent cation concentration. Expression of a recombinant intracellular domain of the calcium channel (Lc753–893) abolished evoked release in the reconstituted assay. Also, mutations at the synaptotagmin C2A polylysine motif, a channel interaction site, abolished depolarization-evoked capacitance transients, consistent with release studies in PC12 cells. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic and nonsynaptic exocytosis and examine the role of other proteins implicated in these processes.

Keywords

Ca2+ channels capacitance Cav1.2 evoked release excitosome exocytosis reconstitution synaptic transmission synaptotagmin syntaxin 
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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Roy Cohen
    • 1
  • Bernhard M. Schmitt
    • 2
  • Daphne Atlas
    • 1
  1. 1.Department of Biological ChemistryThe Hebrew University of JerusalemJerusalemIsrael
  2. 2.Department of PhysiologyUniversity of OtagoOtagoNew Zealand

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