The trans-SNARE-regulating function of Munc18-1 is essential to synaptic exocytosis.

Chong Shen,Rui Hua,Haijia Yu,Daniel R Gulbranson,Nathan E Schoppa,Chen Zhang,Jingshi Shen,Shailendra S Rathore

doi:10.1038/ncomms9852

Abstract

The fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane requires two classes of molecules-SNAP receptor (SNARE) and Sec1/Munc18 (SM) protein. Reconstitution studies suggest that the SM protein Munc18-1 promotes the zippering of trans-SNARE complexes and accelerates the kinetics of SNARE-dependent membrane fusion. However, the physiological role of this trans-SNARE-regulating function in synaptic exocytosis remains to be established. Here we first demonstrate that two mutations in the vesicle-anchored v-SNARE selectively impair the ability of Munc18-1 to promote trans-SNARE zippering, whereas other known Munc18-1/SNARE-binding modes are unaffected. In cultured neurons, these v-SNARE mutations strongly inhibit spontaneous as well as evoked neurotransmitter release, providing genetic evidence for the trans-SNARE-regulating function of Munc18-1 in synaptic exocytosis. Finally, we show that the trans-SNARE-regulating function of Munc18-1 is compromised by a mutation associated with Ohtahara Syndrome, a severe form of epilepsy.

Highlights

The fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane requires two classes of molecules—SNAP receptor (SNARE) and Sec1/Munc[18] (SM) protein
We previously showed that two motifs on VAMP2—S61/E62 and S75/Q76—are required for the stimulation of lipid mixing by Munc[] (Fig. 1a)[32]
Merging of the labelled VAMP2 liposomes with unlabelled t-SNARE liposomes led to the dilution and dequenching of sulforhodamine B fluorescence

Summary

Introduction

The fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane requires two classes of molecules—SNAP receptor (SNARE) and Sec1/Munc[18] (SM) protein. Reconstitution studies suggest that the SM protein Munc[] promotes the zippering of trans-SNARE complexes and accelerates the kinetics of SNARE-dependent membrane fusion. We first demonstrate that two mutations in the vesicle-anchored v-SNARE selectively impair the ability of Munc[] to promote trans-SNARE zippering, whereas other known Munc18-1/SNARE-binding modes are unaffected. In cultured neurons, these v-SNARE mutations strongly inhibit spontaneous as well as evoked neurotransmitter release, providing genetic evidence for the trans-SNARE-regulating function of Munc[] in synaptic exocytosis. Critical to the regulation of SNARE assembly[29,30], this syntaxin-binding activity is not conserved among SM family proteins and is thought to represent an exocytosis-specific role of Munc[] Characterized by two or three charged residues followed by a hydrophobic leucine or phenylalanine residue, the N-peptide motif binds to a peripheral pocket on its cognate SM protein[36,37]

Methods

Results

Conclusion