Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium in¯ux is thought to control this process via Ca 2+ -binding proteins that associate with components of the SNARE complex. Ca 2+ /calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP 77±90 ), and residues involved were identi®ed by plasmon resonance spectroscopy. Microinjection of wild-type VAMP 77±90 , but not mutant peptides, inhibited catecholamine release from chromaf®n cells monitored by carbon ®bre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca 2+ -dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q 76 V, F 77 W) VAMP, but additional targeted mutations in VAMP 77±90 abolished its ability to rescue release. The calmodulin-and phospholipid-binding domain of VAMP 2 is thus required for Ca 2+ -dependent exocytosis, possibly to regulate SNARE complex assembly. Keywords: neuroendocrine cells/secretory vesicle/ SNARE/tetanus toxin IntroductionNeurones and neuroendocrine cells release transmitters and neuropeptides by calcium-dependent exocytosis of the contents of vesicles docked at the plasma membrane. This process requires assembly of trimeric SNARE complexes formed by the vesicle-associated membrane protein synaptobrevin (VAMP 2) and two partners that are expressed mainly in the plasma membrane, syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25) (reviewed by Jahn and Sudhof, 1999;Lin and Scheller, 2000;Mayer, 2001). Analysis of a minimal complex composed uniquely of the interacting domains of these three proteins has revealed a parallel bundle of four a-helices (one from VAMP 2, one from syntaxin 1 and two from SNAP-25) twisted into a superhelical structure (Sutton et al., 1998). Extrapolation of these data to a situation in which VAMP 2 and syntaxin 1/SNAP-25 are anchored in distinct lipid bilayers (i.e. docked vesicle membranes and plasma membranes, respectively) led to a proposal for trans SNARE complex function. The zippingup of SNARE complexes from the N-terminus to the C-terminus would pull the opposing C-terminal transmembrane anchors towards each other and promote membrane fusion at the vesicle±plasma membrane interface.Abundant evidence from the use of botulinum and tetanus toxins (BoNT and TeNT, respectively), which inhibit transmitter release by cleaving SNARE proteins (Xu et al., 1998;Hua and Charlton, 1999), as well as mutagenesis in invertebrates and mice (Fergestad et al., 2001;Schoch et al., 2001;Washbourne et al., 2002), have consolidated the view that SNARE proteins are required for exocytosis. However, the precise role of SNARE complex assembly in membr...

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...

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) protein interactions at the synaptic vesicle͞ plasma membrane interface play an essential role in neurotransmitter release. The membrane-proximal region (amino acids 77-90) of the v-SNARE vesicle-associated membrane protein 2 (VAMP 2, synaptobrevin) binds acidic phospholipids or Ca 2؉ ͞calmodulin in a mutually exclusive manner, processes that are required for Ca 2؉ -dependent exocytosis. To address the mechanisms involved, we asked whether this region of VAMP can interact with cis (outer vesicle leaflet) and͞or trans (inner plasma membrane leaflet) lipids. To evaluate cis lipid binding, recombinant VAMP was reconstituted into liposomes and accessibility to site-directed antibodies was probed by surface plasmon resonance. Data indicated that the membrane-proximal domain of VAMP dips into the cis lipid bilayer, sequestering epitopes between the tetanus toxin cleavage site and the membrane anchor. These epitopes were unmasked by VAMP double mutation W89A, W90A, which abolishes lipid interactions. To evaluate trans lipid binding, VAMP was reconstituted in cis liposomes, which were then immobilized on beads. The ability of VAMP to capture protein-free 3 H-labeled trans liposomes was then measured. When cis lipid interactions were eliminated by omitting negatively charged lipids, trans lipid binding to VAMP was revealed. In contrast, when cis and trans liposomes both contained acidic headgroups (i.e., approximating physiological conditions), cis lipid interactions totally occluded trans lipid binding. In these conditions Ca 2؉ ͞calmodulin displaced cis inhibition, transferring the lipid-binding domain of VAMP from the cis to the trans bilayer. Our results suggest that calmodulin acts as a unidirectional Ca 2؉ -activated shuttle that docks the juxtamembrane portion of the v-SNARE in the target membrane to prepare fusion.

Trans SNARE complex assembly is an essential step in Ca 2+ -dependant membrane fusion, although the SNARE proteins do not bind Ca 2+ions. Studies to evaluate how the Ca 2+ sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site ( 76 Q-F 77 ) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca 2+ /calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca 2+ /calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca 2+ -dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q 76 V,F 77 W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K 83 A,K 87 V or W 89 A,W 90 A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca 2+ -dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca 2+ /calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane.