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Molecular Identification and Reconstitution of Depolarization-Induced Exocytosis Monitored by Membrane Capacitance

Roy Cohen ^*, Bernhard M. Schmitt  and Daphne Atlas ^*

^* Department of Biological Chemistry, The Institute of Life Sciences and the Otto Loewi Center, The Hebrew University of Jerusalem, Jerusalem, Israel; and Department of Physiology, University of Otago, Dunedin, New Zealand

Correspondence: Address reprint requests to Daphne Atlas, Dept. of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 919104 Israel. Tel.: 972-26584506; Fax: 972-2-658-5413; E-mail: datlas{at}vms.huji.ac.il.

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.

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