A molecular model to reconstitute depolarization-induced exocytosis by capacitance
Roy Cohen 1, Bernhard Schmitt 2 and Daphne Atlas 1*
1 The Hebrew University
2 Wurrzburg University
* To whom correspondence should be addressed. E-mail: datlas{at}vms.huji.ac.il.
Submitted on April 14, 2005
Revised on June 8, 2005
Accepted on 15 August 2005
 |
Abstract |
|---|
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 sec) 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+ vs. Ba2+) or channel (Lc-, N-type), and depended non-linearly 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.
Key Words:
Ca2+ channels, Exocytosis, capacitance, synaptotagmin, syntaxin, vesicles
