Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.
Bottom Line: Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association.In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions.Our results suggest that Munc18a primarily acts at the prefusion stage.
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.Show MeSH
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Mentions: Reconstitution experiments with a different subset of synaptic proteins showed that Munc18a acts in conjunction with NSF and αSNAP (soluble NSF adaptor protein), locking syntaxin-1A in a closed conformation and setting the stage for ternary trans-neuronal SNARE complex by the action of Munc13 and an approaching vesicle with synaptobrevin-2 (8). In this reconstitution experiment, Munc18a played an essential role to facilitate trans-SNARE complex formation in conjunction with NSF, αSNAP, and Munc13. Although an important step toward understanding the mechanism of Munc18, this study did not provide direct evidence that Munc18a stimulates fusion itself, because the amount of ensemble lipid mixing was roughly the same for a minimal system consisting only of neuronal SNAREs, the C2AB fragment of synaptotagmin-1, and 0.5 μm Ca2+ as well as for the more complete system consisting of the same proteins plus NSF, αSNAP, Munc18a, and Munc13 (see Fig. 4D in Ref. 8). However, it is possible that the ensemble lipid-mixing method used for this particular comparison may have masked an effect of Munc18a by opposing effects on the association of vesicles prior to fusion and fusion itself because ensemble fluorescence assays generally cannot distinguish between these two effects (21).
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.