Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

• DOI: 10.15252/embj.201796484
• Publication Date: 2017-11-18T01:10:37Z
• Authors: Marieke Meijer; Bernhard Dörr; Hanna CA Lammertse; Chrysanthi Blithi...; Jan RT van Weering; Ruud FG Toonen; Thomas H Söllner; Matthijs Verhage
• Keywords: Mice, Knockout; 0303 health sciences; Vesicle-Associated Membrane Protein 2; Articles; Synaptic Transmission; Protein Structure, Secondary; R-SNARE Proteins; Mice; 03 medical and health sciences; Munc18 Proteins; src-Family Kinases; SDG 3 - Good Health and Well-being; SNARE; Munc18-1; Mutation; synaptic transmission; Animals; Synaptic Vesicles; Phosphorylation; priming; SNARE Proteins; Src

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.