Summary
The identity of the proteins that constitute the “minimal molecular machinery” required for depolarization-evoked neurotransmitter release at synapses is still not fully disclosed. Using capacitance monitoring combined with heterologous protein expression in Xenopus oocytes, we were able to reconstitute a fast (<.5 s) secretion that was triggered directly by membrane depolarization. The functional assembly of voltage-gated Ca2+ channel (Cav1.2 or Cav2.2) coexpressed with syntaxin 1A, synaptosome-associated protein of 25kDa (SNAP-25), and synaptotagmin led to the reconstitution of depolarization-evoked secretion. Botulinum C1, botulinum A, and tetanus toxin were used to establish that this minimal set of proteins, named the excitosome complex, was necessary and sufficient for reconstituting depolarization-induced exocytosis. Similar to synaptic transmission, the capacitance changes were sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channels (Lc or N type; ionotropic glutamate GLUR3), and depended nonlinearly on extracellular divalent cation concentration. Expression of a recombinant intracellular domain of the calcium channel (Lc753–893) abolished evoked release in the reconstituted assay. Also, mutations at the synaptotagmin C2A polylysine motif, a channel interaction site, abolished depolarization-evoked capacitance transients, consistent with release studies in PC12 cells. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic and nonsynaptic exocytosis and examine the role of other proteins implicated in these processes.
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Cohen, R., Schmitt, B.M., Atlas, D. (2008). Reconstitution of Depolarization and Ca2+-Evoked Secretion in Xenopus Oocytes Monitored by Membrane Capacitance. In: Ivanov, A.I. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 440. Humana Press. https://doi.org/10.1007/978-1-59745-178-9_21
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DOI: https://doi.org/10.1007/978-1-59745-178-9_21
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