Molecular Mechanisms of Fast Neurotransmitter Release

Abstract

This review summarizes current knowledge of synaptic proteins that are central to synaptic vesicle fusion in presynaptic active zones, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), synaptotagmin, complexin, Munc18 (mammalian uncoordinated-18), and Munc13 (mammalian uncoordinated-13), and highlights recent insights in the cooperation of these proteins for neurotransmitter release. Structural and functional studies of the synaptic fusion machinery suggest new molecular models of synaptic vesicle priming and Ca²⁺-triggered fusion. These studies will be a stepping-stone toward answering the question of how the synaptic vesicle fusion machinery achieves such high speed and sensitivity.

Keywords: Ca2+ triggering; action potential; fusion protein; synaptic vesicle fusion; synaptic vesicle priming.

Figure 1.. The roles of SNAREs in the synaptic vesicle cycle.

SNAREs form a trans complex that juxtaposes membranes after synaptic vesicle docking and priming. In combination with a Ca²⁺-sensor, evoked fusion occurs upon an action potential. During fusion, SNARE complexes are fully formed (cis complex). Cis SNARE complexes are disassembled by the ATPase NSF in conjunction with SNAPs and made available for another round of synaptic vesicle formation.

Figure 2.. Single-vesicle fusion assay.

(a) The schema shows the initial setup with proteoliposomes with reconstituted synaptobrevin-2 and Syt1 that mimic synaptic vesicles (SV vesicles) and surface-tethered proteoliposomes with reconstituted syntaxin-1A and SNAP-25A that mimic the plasma membrane (PM vesicles). Cpx1 is included in solution. A soluble dye is encapsulated into the SV vesicles, and in some experiments, a lipid dye is incorporated into the SV vesicle membrane. These dyes are at high enough concentration to result in self-quenching of the fluorescence intensity. An increase in volume or surface area, respectively, will produce an increase in the respective fluorescence intensity. (b) Ca²⁺-independent fusion is observed for SV vesicles that are associated with tethered PM vesicles. (c) Ca²⁺-triggered fusion events are observed. For more details see references (, –80, 82).

Figure 3.. Reconstitution with neuronal SNAREs, Cpx1, Syt1, NSF, αSNAP, Munc18, Munc13.

(a) Prior to the schema shown in Fig. 2a, a step is added that dissociates the syntaxin-1A/SNAP-25A complex by NSF, αSNAP, ATP, Mg²⁺ and concomitant formation of the syntaxin-1A/Munc18 complex. Incubation of the resulting syntaxin-1A/Munc18 vesicles is performed in the presence of Munc13–1, Cpx1, and SNAP-25A. (b) Ca²⁺-sensitivity of the fusion assay using the schema shown in panel a. For more details see (80).

Figure 4.. Munc13 and Munc18 assist in proper SNARE complex formation.

Starting from the syntaxin-1A/Munc18 complex, ternary SNARE complex is formed upon addition of the MUN domain of Munc13, SNAP-25A, and synaptobrevin-2. Single-molecule FRET measurements involving pairs of fluorescent dyes indicate that the components of the SNARE complex are parallel with respect to each other. For more details see (80).

Figure 5.. The primary SNARE/Syt1 interface is structurally conserved.

Superposition of the primary interface observed in crystal structures of the Ca²⁺ (3.5 Å resolution, PDB code 5CCG, color) and Mg²⁺ (4.1 Å resolution, PDB code 5CCI, gray/white) bound structures of the SNARE/Syt1 (184), and of the SNARE/Cpx1/Syt1 complex (1.8 Å resolution, PDB code 5W5C, black) (185). Note, that the crystal packing of the SNARE/Cpx1/Syt1 complex is very different from that of the SNARE/Syt1 complexes (185).

Figure 6.. Structure of the SNARE/Cpx1/Syt1 complex.

Shown are both the primary and the tripartite interface. For details see (185).

Figure 7.. Point contact between proteoliposomes with neuronal SNAREs, Cpx1, Syt1, and Munc13.

(a) Shown is a slice of an isosurface representation of a SV vesicle (bottom) and a PM vesicle (top) in the presence of the C1C2BMUN fragment of Munc13. (b) Close-up view of grey scale tomographic 2D-slices of the contact site between the two membranes. For more details see (44).

Figure 8.. Model of primed pre-fusion SNARE/Cpx1/Syt1 complexes.

In this model, two SNARE/Cpx1/Syt1 complexes (185) form a contact between a synaptic vesicle (top) and the plasma membrane (bottom). Blue: synaptobrevin-2, green: SNAP-25A, red: syntaxin-1A, yellow: Cpx1, light/dark magenta: Syt1s. The molecules and membranes are drawn to scale, with a ~ 40 Å separation between membranes as observed by cryoET (44). Note that the relative arrangement of the two SNARE/Cpx1/Syt1 complexes is unknown. Also, there can be more than two such complexes forming long membrane contacts (44).