SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.

Abstract

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca2+-signaled exocytosis is effectively blocked by antibodies and peptides to a Several lines of evidence have identified three synaptic proteins, syntaxin, VAMP, and SNAP-25, which together with synaptotagmin form the core complex postulated to coordinate regulated vesicular fusion for neurotransmitter release (see refs. 3-5). In vitro studies with solubilized and recombinant proteins have begun to define the molecular relationships between these proteins (6-9). In an initial 7S complex, SNAP-25 binds both syntaxin 1A and VAMP to increase the strength and specificity of their interaction (8, 9). Binding of a-SNAP to this complex displaces synaptotagmin from syntaxin and provides entry of N-ethylmaleimide-sensitive fusion protein (NSF) to form a 20S prefusion complex that upon ATP hydrolysis subsequently leads to its disassembly (6). These observations have led to the proposal that neurotransmission represents a specialization of general membrane trafficking in which recognition between neural-specific vesicle-and targetsoluble NSF attachment protein receptors (v-and t-SNAREs) targets synaptic vesicles to the plasma membrane and the ATP hydrolysis step mediated by NSF to drive exocytosis (10).These in vitro studies, however, have yet to resolve the full roles played by these synaptic vesicle and plasma membrane proteins in the cascade of events required for neurotransmitter release. For

Conserved Prefusion Protein Assembly in Regulated Exocytosis

“…The calcium-triggered interaction is less specific, with SYT binding individual SNAREs, regardless of whether they are assembled or not (Bai et al, 2004), and also non-SNARE proteins, e.g., tubulin (Honda et al, 2002). Importantly, in the context of native synaptic membranes, a calcium-dependent dissociation of SYT from the SNAREs has been observed, stressing an important role for the constitutive SYT/SNARE association (Mehta et al, 1996;Leveque et al, 2000).…”

“…SYT possesses two C2 domains, C2A and C2B, both being capable of calciumdependent phospholipid binding in in vitro reactions (Davletov and Sudhof, 1993;. The original observed property of native SYT, however, was its ability to copurify with assembled SNAREs, in the absence of calcium, from brain extracts (Sollner et al, 1993;Mehta et al, 1996;Leveque et al, 2000). The ability of the assembled, rather than individual, t-SNARE proteins to bind SYT and synaptobrevin may be a reflection of large structural changes in syntaxin and SNAP-25 upon their interaction (Fasshauer et al, 1997).…”

“…The calcium-triggered interaction is less specific, with SYT binding individual SNAREs, regardless of whether they are assembled or not (Bai et al, 2004), and also non-SNARE proteins, e.g., tubulin (Honda et al, 2002). Importantly, in the context of native synaptic membranes, a calcium-dependent dissociation of SYT from the SNAREs has been observed, stressing an important role for the constitutive SYT/SNARE association (Mehta et al, 1996;Leveque et al, 2000).The constitutive SYT coupling to the syntaxin/SNAP-25 assembly may be sufficient to provide an organized scaffold for a calcium-enhanced collision of nascent SNARE ␣-helices, resulting in rapid formation (Myers and Oas, 2001) of the fusogenic ternary SNARE complex. The proposed model highlights the pivotal role for C2B in linking the vesicular and the plasma membrane fusion machineries before calcium-triggered events and is in agreement with available in vivo findings.…”

Localization and Function of SolubleN-Ethylmaleimide-sensitive Factor Attachment Protein-25 and Vesicle-associated Membrane Protein-2 in Functioning Gastric Parietal Cells

“…The cleavage of SNAREs by Clostridial neurotoxins has been established as an essential tool to investigate the role of these proteins in neurotransmission (21,22). The C terminus of SNAP-25 that contributes one helix to the four-helix cytoplasmic bundle is believed to be critical for SNARE complex formation and has been implicated in the final step of calciumtriggered exocytosis (4,(23)(24)(25)(26)(27). More specifically, botulinum neurotoxins A and E cleave the C-terminal domain of SNAP-25, causing complete or partial inhibition of secretion in neuronal and endocrine systems as well as pancreatic acinar cells (22,28).…”

Chronic Antidepressants Induce Redistribution and Differential Activation of αCaM Kinase II between Presynaptic Compartments

“…Second, antidepressants induced a redistribution of aCaM kinase II between synaptic membranes and synaptic vesicles of HC. Based on previous studies, it can be argued that the presence of CaM kinase II in synaptic membranes is mainly due to the presence of a pool of vesicles docked to the plasma membrane (Mehta et al, 1996). This morphologically defined pool of vesicles has been correlated with the functionally defined readily releasable pool (RRP), containing the vesicles that fuse with the membrane and release glutamate upon stimulation (Schikorski and Stevens, 2001).…”