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: Phosphorylation of Munc18a greatly reduces interaction with syntaxin-1A (41), and a phosphorylation-mimicking mutant Unc-18(S322E) of Caenorhabditis elegans Unc-18 (S322E) reduces binding to syntaxin-1A. However, the role of phosphorylation in neurons was found to be relatively subtle; the two phosphorylation sites in Munc18a appear to be important for post-tetanic potentiation (59). In our system, similar to wild type Munc18a, the phosphorylation mimic mutant of Munc18a(S306E/S313E) 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. The phosphorylation mimic Munc18a(S306E/S313E) also reduced vesicle association but to a lesser degree (Fig. 7A). The lessened effect on SV-PM vesicle association by the phosphorylation mimic is correlated with its reduced affinity to syntaxin-1A compared with wild type Munc18a, as determined by bio-layer interferometry experiments (Fig. 9). Thus, these experiments further corroborate that the sequestration of syntaxin-1A by Munc18a is related to the interaction between Munc18a and syntaxin-1A.
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.