TY - JOUR
T1 - Molecular identification and reconstitution of depolarization-induced exocytosis monitored by membrane capacitance
AU - Cohen, Roy
AU - Schmitt, Bernhard M.
AU - Atlas, Daphne
PY - 2005/12
Y1 - 2005/12
N2 - 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 1 A, 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 Sr 2+) 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.
AB - 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 1 A, 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 Sr 2+) 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.
UR - http://www.scopus.com/inward/record.url?scp=28444493051&partnerID=8YFLogxK
U2 - 10.1529/biophysj.105.064642
DO - 10.1529/biophysj.105.064642
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C2 - 16150968
AN - SCOPUS:28444493051
SN - 0006-3495
VL - 89
SP - 4364
EP - 4373
JO - Biophysical Journal
JF - Biophysical Journal
IS - 6
ER -