Molecular machines governing exocytosis of synaptic vesicles

Abstract

Calcium-dependent exocytosis of synaptic vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine.

Figure 1

Trafficking pathways in the nerve terminal. Synaptic vesicles are filled with neurotransmitter and stored in the cytoplasm. Active vesicles are translocated to release sites in the active zone where they dock. Priming involves all steps required to acquire release readiness of the exocytotic complex. Although usually assumed to occur after docking, priming and even triggering may precede docking during sustained activity, resulting in immediate fusion of an arriving vesicle. After exocytosis, the vesicle proteins probably remain clustered and are then retrieved by endocytosis. Despite some lingering controversies, consensus is emerging that retrieval is generally mediated by clathrin-mediated endocytosis. After clathrin uncoating, synaptic vesicles are regenerated within the nerve terminal, probably involving passage through an endosomal intermediate. Actively recycling vesicles are in slow exchange with the reserve pool. See text for more details

Figure 2

Schematic depictions of domain structures and crystal structures of core proteins of the neuronal fusion machine. The dashed lines between the N-peptide (N) and Habc domain represent flexible regions in syntaxin. For synaptotagmin the two Ca²⁺ binding sites are indicated. Note that the domain structure of the large multi-domain protein Munc13 is shown five times smaller than those of the other domain structures. A high-resolution structure was obtained for the C-terminal half of the MUN domain. See text for details. The data for structures are from: Munc13-1 (C and D subdomains) (48),Synaptotagmin 1 (C2A and C2B domain) (117), Munc18-1 (blue-green, in complex with syntaxin (red)) (32),Habc domain (16), SNARE complex (18),complexin (63). aa, amino acid.

Figure 3

Alternative models describing the steps between priming and fusion. Priming I involves arrest of a partially zippered SNARE complex, here shown with bound Munc18, Munc13 and synaptotagmin. Calcium influx triggers binding of synaptotagmin to the SNARE complex and to the plasma membrane (involving PI(4,5)P₂, not shown here), associated with displacement of complexin and (possibly) Munc18 and/or Munc13. Priming II involves arrest after positioning of the vesicle with the aid of active zone components and (possibly) contact of synaptotagmin with PI(4,5)P₂ in the plasma membrane, but no contact between the SNAREs. Ca²⁺-triggering pulls the vesicle closer via synaptotagmin-mediated cross-linking, resulting in SNARE assembly, associated with full opening of syntaxin and displacement of Munc18, and binding of complexin. See text for details.

Figure 4

Transition states during membrane fusion. Intermediates of the fusion pathway. The top drawings represent intermediate states of the membrane along the pathway as predicted by the elastic theory. Below, snapshots of intermediate states of a simulation of SNARE-mediated fusion are shown, which, although roughly corresponding to the elastic model, differ in detail and in their energy predictions (adapted from ref. , courtesy of J. Rissellada and H. Grubmüller).