Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with genetically-engineered photopharmacology.

Damien Lemoine,Bertrand Lambolez,Jérome Tange,Ludovic Tricoire,Alexandre Mourot,Sarah Mondoloni,Antoine Taly,Philippe Faure

doi:10.7554/elife.59026

Abstract

Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, yet they don't bind glutamate and are considered orphan. Progress in defining the ion channel function of GluDs in neurons has been hindered by a lack of pharmacological tools. Here, we used a chemo-genetic approach to engineer specific and photo-reversible pharmacology in GluD2 receptor. We incorporated a cysteine mutation in the cavity located above the putative ion channel pore, for site-specific conjugation with a photoswitchable pore blocker. In the constitutively open GluD2 Lurcher mutant, current could be rapidly and reversibly decreased with light. We then transposed the cysteine mutation to the native receptor, to demonstrate with high pharmacological specificity that metabotropic glutamate receptor signaling triggers opening of GluD2. Our results assess the functional relevance of GluD2 ion channel and introduce an optogenetic tool that will provide a novel and powerful means for probing GluD2 ionotropic contribution to neuronal physiology.

Highlights

The delta glutamate receptors, GluD1 and GluD2, belong to the ionotropic glutamate receptor family, yet they don’t bind glutamate (Yuzaki and Aricescu2017)
They are considered as glutamate receptors solely based on their strong sequence and structure homology with AMPA, NMDA and kainate receptors (Lomeli et al 1993; Araki et al 1993; Schmid and Hollmann 2008; Elegheert et al 2016; Burada, Vinnakota and Kumar 2020a; Burada, Vinnakota and Kumar 2020b)
In darkness or under green light (500-535 nm), the photoswitchable tethered ligand (PTL) adopts an elongated shape and reaches the lumen, resulting in ion channel blockade, while under violet light (380-390 nm), it switches to a twisted, shorter configuration, relieving blockade

Summary

Introduction

The delta glutamate receptors, GluD1 and GluD2, belong to the ionotropic glutamate receptor (iGluR) family, yet they don’t bind glutamate Deletion of genes coding for GluD1 or GluD2 in mouse results in marked behavioral alterations (Yadav et al.2012; Lalouette et al 2001; Yadav et al 2013; Nakamoto, Kawamura, et al 2020), and neurodevelopmental and psychiatric diseases (Griswold et al 2012; Treutlein et al.2009; Greenwood et al 2011; Cristino et al 2019), attesting to their functional importance in brain circuits Despite their structural similarity with other iGluRs, and notably the presence of a ligand binding domain (LBD), GluDs stand out because they are not activated by glutamate (Araki et al 1993; Lomeli et al 1993). Pore blockers for GluDs, such as pentamidine and 1-Naphthyl acetyl spermine (NASPM), were previously identified using a point mutation (A654T) in GluD2 that confers constitutive ion flow and causes the degeneration of cerebellar PCs in Lurcher (GluD2Lc) mice (Wollmuth et al 2000; Zuo et al 1997) These molecules are pore blockers of NMDA and AMPA receptors, respectively. This novel tool, called lightcontrollable GluD2 (LiGluD2), allows rapid, reversible and pharmacologically-specific control of ionic current through GluD2, and may help provide a mechanistic understanding of how this receptor contributes to brain circuit function and behaviors

Results

Discussion

Materials and methods