Molecular identification and reconstitution of depolarization-induced exocytosis monitored by membrane capacitance.
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.
Cohen
Roy
R
Department of Biological Chemistry, The Institute of Life Sciences and the Otto Loewi Center, The Hebrew University of Jerusalem, Jerusalem, Israel.
Schmitt
Bernhard M
BM
Atlas
Daphne
D
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