Munc18a Does Not Alter Fusion Rates Mediated by Neuronal SNAREs, Synaptotagmin, and Complexin

Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca2+-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). 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. Background: Munc18-1 is an important factor for synaptic transmitter release, but its molecular mechanism remains an enigma. Results: Munc18a does not affect fusion kinetics. It can sequester syntaxin-1A molecules from the syntaxin-1A·SNAP-25 t-SNARE complex. Conclusion: Munc18a has no effect on complete fusion in conjunction with synaptotagmin-1, complexin-1, and neuronal SNAREs. Significance: This work provides new insights into Munc18-1 and its interactions with other synaptic proteins.