A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency.

Xiao Min Zhang,Alexis Pierre Bemelmans,Véronique Bernard,Mona Maged,Etienne Herzog,Urielle François,Sonja M Wojcik,Stéphanie Pons,Kätlin Silm,Maria Florencia Angelo,Maria Victoria Fernandez-Busch,Salah El Mestikawy,Yann Humeau,Uwe Maskos,Melissa Deshors,Fabrice P Cordelières,Christelle Martin

doi:10.7554/elife.50401

Abstract

Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active-zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology three and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release.

Highlights

Synaptic vesicles (SVs) engage in multiple protein interactions at the presynaptic active zone [1] and fuse with the presynaptic plasma membrane upon calcium influx, to release their neurotransmitter content [2]
Mammalian VGLUT1 displays a unique double proline-rich (PRD1 530-540; PRD2 550-556) pattern conserved in all mammals and absent in VGLUT2 and -3 or in invertebrate orthologs of VGLUT1 [38]
A remaining spontaneous activity was found in the knock-out cultures that can be attributed to a minor but significant expression of VGLUT2 in hippocampal neurons [35,58,59]. To minimize this contribution we monitored VGLUT2 levels in the culture at several ages and established that the lower plateau is reached between DIV17 and DIV22 when we performed our imaging and patch clamp experiments

Summary

Introduction

Synaptic vesicles (SVs) engage in multiple protein interactions at the presynaptic active zone [1] and fuse with the presynaptic plasma membrane upon calcium influx, to release their neurotransmitter content [2]. FRAP of VGLUT1venus fluorescence revealed that VGLUT1 overexpression further reduces SV exchange with axonal pools compared to the rescue of the knock-out to endogenous levels (Figure 1E). Structure analysis of VGLUT1 To uncover the molecular mechanism by which VGLUT1 regulates SV super-pool size, we generated a series of mutants spanning the sequence of the transporter (Figure 2A and Table 1).

Results

Conclusion