Reconstitution of depolarization and Ca2+-evoked secretion in Xenopus oocytes monitored by membrane capacitance.
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 25 kDa (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.
Cohen
Roy
R
Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel.
Schmitt
Bernhard M
BM
Atlas
Daphne
D
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002412:Cations / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D025802:Electric Capacitance; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D018014:Gene Transfer Techniques; D008564:Membrane Potentials; D009498:Neurotoxins / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D009435:Synaptic Transmission / Q000187:drug effects; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014982:Xenopus laevis
D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D002412:Cations; C419385:L-type calcium channel alpha(1C); D009498:Neurotoxins; D018377:Neurotransmitter Agents; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1