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: Munc18a had no effect on the normalized Ca2+-triggered fusion histograms (Fig. 1, A–F), the synchronization time constant (Fig. 1G), or the probability of spontaneous fusion (Fig. 1H), regardless of whether full-length Stx1a or the N-terminally truncated Stx1a(10–288) fragment were used. Moreover, there was no effect if Munc18a was incubated concurrently when adding free SV vesicles to the immobilized PM vesicles (Fig. 2). We note that the histograms were normalized with respect to the total number of fusion events, so they measure the effect of Munc18a on the intrinsic spontaneous and Ca2+-triggered fusion kinetics of associated vesicle-vesicle pairs. The ability to measure intrinsic fusion kinetics is an advantage compared with ensemble fusion experiments, where effects on docking and fusion cannot be separated (21).
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.