Reconstitution of Depolarization and Ca2+-Evoked Secretion in Xenopus Oocytes Monitored by Membrane Capacitance

Abstract

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.

Conformational Changes Induced in Voltage-gated Calcium Channel Cav1.2 by BayK 8644 or FPL64176 Modify the Kinetics of Secretion Independently of Ca2+ Influx

“…Secretion has previously been shown to be mediated by releasing a competent excitosome complex assembled by VGCC and synaptic proteins (32, 34 -36, 38, 39, 56 -61). The frequent channel opening induced by BayK and FPL (17,18) implies potentially more interactive channels, frequent encounters of the channel with synaptic proteins, and generation of more competent releasing complexes (35,36,38,57,60,61). Therefore, the increase in the frequency of channel opening by BayK and FPL could have accounted in part for the increase in the rate of secretion observed in all the cations.…”

Massive surface membrane expansion without involvement of classical exocytic mechanisms

“…It has long been known that diverse cell types can expand their PM by substantial amounts in response to large elevations of cytoplasmic Ca 2+ (Coorssen et al, 1996;Ninomiya et al, 1996;Kasai et al, 1999;Togo et al, 2003;Kreft et al, 2004;Bretscher, 2008;Cohen et al, 2008;Yaradanakul et al, 2008). This expansion has often been suggested to mediate repair of membrane wounds (Togo et al, 2003;Bradke et al, 2012;Cooper and McNeil, 2015;Corrotte et al, 2015), and an involvement of SNARE-mediated mechanisms seems well established in some cell wounding protocols (Bi et al, 1995).…”

“…It is long been known that many cell types can expand their surface membrane by a large amount in response to elevations of cytoplasmic Ca (Coorssen et al, 1996;Ninomiya et al, 1996;Kasai et al, 1999;Togo et al, 2003;Kreft et al, 2004;Bretscher, 2008;Cohen et al, 2008;Yaradanakul et al, 2008), and this expansion has been suggested to play a role in the response of cells to membrane wounding (Corrotte et al, 2015). In the accompanying article (al., XXXX) we have shown that TMEM16F initiates these responses to cytoplasmic Ca elevation.…”

“…Both electrical and optical methods have revealed that diverse cell types increase their PM area substantially during large elevations of cytoplasmic Ca 2+ (Coorssen et al, 1996;Ninomiya et al, 1996;Kasai et al, 1999;Togo et al, 2003;Kreft et al, 2004;Bretscher, 2008;Cohen et al, 2008;Yaradanakul et al, 2008). PM expansion can double PM area within a few seconds in some cell types Yaradanakul et al, 2008;Bricogne, XXXX).…”

The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

“…In these cells, the channel is an integral part of the signaling complex that triggers secretion [29–32]. Voltage-driven perturbations of a Ca 2+ -bound channel were suggested to transmit the signal from the channel to the exocytotic machinery and trigger fast release of vesicles assembled with the channel [28, 31, 33–35]. Therefore, channel phosphorylation would be expected to modify secretion.…”