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

Abstract

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.

A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity

“…This study shows that T479 is among several phosphorylation sites in Munc18-1 that are constrained and can be phosphorylated in vitro. Despite other established strong effects of Munc18-1 phosphorylation 5,[16][17][18]42,43 , T479 is not a major regulatory site under the conditions tested here. Synaptic transmission is maintained at wildtype level when mimicking or preventing Dyrk1a-dependent phosphorylation, except for recovery after intense stimulation.…”

“…This region is not associated with binding to synaptobrevin/VAMP2 or syntaxin 44,45 , the canonical interactors of Munc18-1, which are essential for its function. A phosphorylation site that critically influences Munc18-1 function, the Src kinase phosphorylation site (Y473) 17 , is located close to T479. Furthermore, Y473 is a predicted binding site of synaptobrevin/VAMP2 17,45 .…”

“…A phosphorylation site that critically influences Munc18-1 function, the Src kinase phosphorylation site (Y473) 17 , is located close to T479. Furthermore, Y473 is a predicted binding site of synaptobrevin/VAMP2 17,45 . Despite being 6 amino acids away from Y473, mimicking or preventing phosphorylation at T479 does not have a strong regulatory effect on synaptic transmission and is therefore likely to be located away from where synaptobrevin/VAMP2 and Munc18-1 interact.…”

“…Using whole-cell patch-clamp electrophysiology on single hippocampal neurons grown on glia islands (autapses), we investigated the effects of M18 T479A and M18 T479D on synaptic transmission and short-term plasticity using M18 WT as a positive control. Expression of M18 Y473D , mimicking phosphorylation by Src, was previously shown to severely inhibit synaptic transmission 17 and was used here as a negative control to test the successful conditional deletion of Munc18-1. Neurons expressing M18 T479A or M18 T479D showed a similar spontaneous mEPSC frequency ( Fig.…”

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1

“…Hippocampi Autaptic hippocampal cultures were prepared as described previously [91]. Briefly, micro-islands were prepared with a solution containing 0.1 mg/ml poly-D-lysine (sigma), 0.7 mg/ml rat tail collagen (BD Biosciences) and 10 mM acetic acid (Sigma) applied with a custom-made rubber stamp (dot diameter 250 μm).…”