title: Molecular identification and reconstitution of depolarization-induced exocytosis monitored by membrane capacitance.
abstract: Regulated exocytosis of neurotransmitters at synapses is fast and tightly regulated. It is unclear which proteins constitute the "minimal molecular machinery" for this process. Here, we show that a novel technique of capacitance monitoring combined with heterologous protein expression can be used to reconstitute exocytosis that is fast (<0.5 s) and triggered directly by membrane depolarization in Xenopus oocytes. Testing synaptic proteins, voltage-gated Ca2+ channels, and using botulinum and tetanus neurotoxins established that the expression of a Ca2+ channel together with syntaxin 1A, SNAP-25, and synaptotagmin was sufficient and necessary for the reconstitution of depolarization-induced exocytosis. Similar to synaptic exocytosis, the reconstituted release was sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channel (Lc-, N-type), and depended nonlinearly on divalent cation concentration. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic exocytosis.
authors.affiliation: Department of Biological Chemistry, The Institute of Life Sciences and the Otto Loewi Center, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Roy
authors.initials: R
authors.lastname: Cohen
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Schmitt
authors.forename: Daphne
authors.initials: D
authors.lastname: Atlas
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000502:physiology*; D002478:Cells, Cultured; D017097:Electric Impedance; D005089:Exocytosis / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D008991:Monitoring, Physiologic / Q000379:methods*; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D013569:Synapses / Q000502:physiology*; D014982:Xenopus laevis