Ca ²⁺ -dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin

Abstract

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca 2؉ could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca 2؉ -dependent calmodulin binding (Kd ‫؍‬ 500 nM) to glutathione Stransferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP 77-94, immediately C-terminal to the tetanus toxin cleavage site (Q76-F77). In isolated synaptic vesicles, Ca 2؉ ͞calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a 35 S-SNAP-25, syntaxin 1 GST-VAMP1-96 complex was inhibited by Ca 2؉ ͞calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP77-94 and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin͞ phospholipid-binding peptide displayed a significant increase in ␣-helical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca 2؉ may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.T ransmitter release at the nerve terminal occurs via calciumdependent exocytosis of the contents of synaptic vesicles at the presynaptic plasma membrane. Synaptic vesicle fusion involves the assembly of a heterotrimeric synaptic core [soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE)] complex composed of the vesicle-associated membrane protein (VAMP 2 or synaptobrevin) and two predominantly plasma membrane proteins, syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) (1, 2). The fundamental importance of these components is underlined by the fact that the metalloprotease activities of the botulinum (BoNT) and tetanus (TeTx) neurotoxins cleave the synaptic SNARE proteins at well-defined sites and potently inhibit transmitter release (3,4). Structural studies have demonstrated that the synaptic SNARE complex forms a four-helical parallel bundle with a superhelical twist (5, 6). It has been proposed that the assembly of this structure in a trans configuration, i.e., at the interface between a docked synaptic vesicle and the plasma membrane, pulls the opposing membranes together and may ultimately drive bilayer fusion (7). In support of this hypothesis, reconstitution of purified v-SNARE and t-SNARE proteins into distinct vesicle populations led to an increase in lipid mixing between the two vesicle pools, indicative of fusion (8). Although assembly is thought to be initiated in a trans configurati...

Determinants of Synaptobrevin Regulation in Membranes

“…We therefore tested whether calmodulin does indeed bind to synaptobrevin in a calcium-dependent manner, and if so, whether binding was preserved when synaptobrevin is reconstituted into liposomes. When calmodulin and synaptobrevin (lacking the transmembrane domain) were mixed, a Ca 2ϩ -dependent increase in tryptophan fluorescence emission, associated with a slight blue shift, was observed ( Figure 4B), thus confirming the previous findings by Quetglas et al (2000). In addition, no effect of Ca 2ϩ /calmodulin on SNARE complex formation was observed (Supplementary Figure S3).…”

“…It has been shown previously that the C-terminal portion of the SNARE motif and the adjacent linker region, residues 77-90, constitutes a Ca 2ϩ -dependent binding site for calmodulin (Quetglas et al, 2000), raising the possibility that calmodulin controls the activity of synaptobrevin. To investigate this issue, we added Ca 2ϩ /calmodulin to a standard liposome fusion reaction, but no change of fusion rates was observed ( Figure 4A).…”

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

“…Hence, the ability of syb2 mutants to support release in vivo reflects the contribution of each corresponding amino acid to the SNARE complex dimer interface. Previous studies have implicated W90 in calmodulin-dependent regulation of exocytosis (Quetglas et al, 2000(Quetglas et al, , 2002. However, because W90A would disrupt the calmodulin binding motif within syb2 (Rhoads and Friedberg, 1997;Chin and Means, 2000), the ability of this mutant to largely support secretion would suggest that such an interaction does not play a major role in vivo.…”

“…In either case, various structural and mutational data Rickman et al, 2006;Lynch et al, 2007) further indicate that residues distinct from those involved in dimerization seem to be responsible for complexin and synaptotagmin binding to SNARE complexes. W89 and W90 of syb2 have been implicated in binding to phospholipids (Quetglas et al, 2000(Quetglas et al, , 2002de Haro et al, 2004). In fact, it has been suggested that the reversible insertion of these residues of syb2 into the synaptic vesicle membrane may decrease the availability of syb2 and hence the probability of SNARE complex formation (Hu et al, 2002;Kweon et al, 2003).…”

SNARE, V-ATPase et neurotransmission

“…Ce même domaine de VAMP2 est connu pour son interaction avec la CaM. De plus, les mêmes déterminants moléculaires de la VAMP2 gouvernent son interaction avec la sous-unité c et la CaM [19]. Effectivement, nous avons aussi montré que l'interaction VAMP2/sousunité c est régulée par la Ca 2+ -CaM.…”