Synaptobrevin 1 mediates vesicle priming and evoked release in a subpopulation of hippocampal neurons

Abstract

Zimmermann J, Trimbuch T, Rosenmund C. Synaptobrevin 1 mediates vesicle priming and evoked release in a subpopulation of hippocampal neurons. J Neurophysiol 112: 1559 -1565, 2014. First published June 18, 2014 doi:10.1152/jn.00340.2014.-The core machinery of synaptic vesicle fusion consists of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, the two t-SNAREs at the plasma membrane (SNAP-25, Syntaxin 1) and the vesicle-bound v-SNARE synaptobrevin 2 (VAMP2). Formation of the trans-oriented four-␣-helix bundle between these SNAREs brings vesicle and plasma membrane in close proximity and prepares the vesicle for fusion. The t-SNAREs are thought to be necessary for vesicle fusion. Whether the v-SNAREs are required for fusion is still unclear, as substantial vesicle priming and spontaneous release activity remain in mammalian mass-cultured synaptobrevin/cellubrevindeficient neurons. Using the autaptic culture system from synaptobrevin 2 knockout neurons of mouse hippocampus, we found that the majority of cells were devoid of any evoked or spontaneous release and had no measurable readily releasable pool. A small subpopulation of neurons, however, displayed release, and their release activity correlated with the presence and amount of v-SNARE synaptobrevin 1 expressed. Comparison of synaptobrevin 1 and 2 in rescue experiments demonstrates that synaptobrevin 1 can substitute for the other v-SNARE, but with a lower efficiency in neurotransmitter release probability. Release activity in synaptobrevin 2-deficient mass-cultured neurons was massively reduced by a knockdown of synaptobrevin 1, demonstrating that synaptobrevin 1 is responsible for the remaining release activity. These data support the hypothesis that both t-and v-SNAREs are absolutely required for vesicle priming and evoked release and that differential expression of SNARE paralogs can contribute to differential synaptic coding in the brain. SNARE; neurotransmitter release; spontaneous release; release probability; short-term plasticity RELEASE OF NEUROTRANSMITTERS in the mammalian brain is mediated by fusion of synaptic vesicles (SVs) with the neuronal plasma membrane. This process has to be both fast and reliable and is performed by evolutionarily conserved protein machinery (Rizo and Rosenmund 2008;Sudhof 2013). Three proteins from the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) family are considered to bring the two membranes in close proximity and carry out membrane fusion (Sollner et al. 1993;Weber et al. 1998). A v-SNARE (anchored in the SV membrane) and the t-SNAREs syntaxin (Bennett et al. 1992) and synaptosomal-associated protein 25 (SNAP-25) (Oyler et al. 1989) (both anchored in the plasma membrane) zipper up from their NH 2 to COOH terminus and thus form a four-␣-helix bundle called the trans-SNARE complex (Poirier et al. 1998;Sorensen et al. 2006;Sutton et al. 1998). Whether fusion of SVs is possible without SNARE proteins is still an open question. The major v-SNARE paralog on SVs i...

Do SNARE Protein Isoforms Determine Fusion Pore Characteristics?

“…Another model proposes a proteinaceous fusion pore, in which the transmembrane domains of the SNARE proteins are incorporated into the pore (Jackson and Chapman, 2008;Fang and Lindau, 2014). The latter hypothesis is supported by previous work by Han et al (2004), who showed that replacing any of three specific amino acids of the syntaxin 1 transmembrane domain with the large amino acid tryptophan altered flux through the fusion pore. These amino acids are located at the same face of the ␣-helix, suggesting that this ␣-helical face lines the fusion pore.…”

“…These amino acids are located at the same face of the ␣-helix, suggesting that this ␣-helical face lines the fusion pore. These data led the authors to propose a model in which the fusion pore consists of a circular arrangement of five to eight syntaxin 1 transmembrane domains (Han et al, 2004). This raises the question of whether the transmembrane domain of synaptobrevin 2 might also be involved in fusion pore formation.…”

“…However, just before this spike, small amounts of cargo are secreted through the fusion pore. This shows up in amperometric measurements as a prespike foot (PSF) (Jackson and Chapman, 2006). For their experiments, Chang et al (2015) used chromaffin cells from synaptobrevin 2 and cellubrevin (VAMP2 and VAMP3, respectively) double knock-out (DKO) mice, in which DCV fusion is almost completely abolished.…”

“…This shows up in amperometric measurements as a prespike foot (PSF) (Jackson and Chapman, 2006). For their experiments, Chang et al (2015) used chromaffin cells from synaptobrevin 2 and cellubrevin (VAMP2 and VAMP3, respectively) double knock-out (DKO) mice, in which DCV fusion is almost completely abolished. Similar to the study by Han et al (2004), 21 mutants of synaptobrevin 2-each with a single amino acid replaced with tryptophan in the transmembrane domain (positions 96 -116)-were individually expressed in the DKO cells.…”

“…For their experiments, Chang et al (2015) used chromaffin cells from synaptobrevin 2 and cellubrevin (VAMP2 and VAMP3, respectively) double knock-out (DKO) mice, in which DCV fusion is almost completely abolished. Similar to the study by Han et al (2004), 21 mutants of synaptobrevin 2-each with a single amino acid replaced with tryptophan in the transmembrane domain (positions 96 -116)-were individually expressed in the DKO cells. Expression of any of these constructs rescued DCV release frequency to wild-type levels (eight times higher than DKO).…”