Tyrosine phosphorylation of Munc18-1 inhibits synaptic transmission by preventing SNAREￂ assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18-1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho-proteomics abolished the stimulatory effect of Munc18-1 on SNARE complex formation ("SNARE-templating") and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18-1-null neurons expressing Munc18-1Y473D. Synaptic transmission was temporarily restored by high-frequency stimulation, as well as by a Munc18-1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non-phosphorylatable Munc18-1 supported normal synaptic transmission. We propose that SFK-dependent Munc18-1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post-docking SNARE-templating role of Munc18-1, resulting in a largely abolished pool of releasable synaptic vesicles. Synopsis This study identifies a novel regulatory site on the presynaptic protein Munc18-1, which phosphorylated state prevents Synaptobrevin2/VAMP2 binding, SNARE-templating and synaptic vesicle priming. Tyrosine phosphorylation of Munc18-1 is a potent way to shut down synaptic transmission. Y473 on Munc18-1 is a substrate for several neuronal members of Src family kinase. A single point mutation (Y473D) designed to mimic tyrosine phosphorylation interfered with Synaptobrevin2/VAMP2 binding and the stimulatory effect of Munc18-1 on SNARE-complex formation. Hippocampal munc18-1 null neurons expressing Munc18-1Y473D are defective in synaptic vesicle priming, but not docking. Synaptic transmission was largely restored by high frequency stimulation or by promoting helix 12 extension in domain 3a of Munc18-1.