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

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.

Delta glutamate receptors are functional glycine- and ᴅ-serine–gated cation channels in situ

“…Ionotropic activity has also been observed in GluD2 upon activation of G q -coupled metabotropic glutamate receptor with glutamate ( 16 ). The constitutive activity of GluD2 A654T Lurcher mutant and activation via the metabotropic glutamate receptor pathway has been shown to be blocked by a photoactivatable channel blocker attached to a cysteine near the channel pathway, further confirming the two pathways to be mediated by the GluD2 receptor channel ( 17 ). Because the agonist-binding and transmembrane domains of glutamate receptors can function independently, the lack of agonist-induced currents in wild-type delta receptors suggests that conformational changes in the agonist-binding domains cannot be transmitted to the transmembrane segments that form the channel pore.…”

Photopharmacology of Ion Channels through the Light of the Computational Microscope

“…Photoswitchable ligands targeting ionotropic glutamate receptors are not limited to PCLs and PTLs binding to the LBD. A recent study [ 122 ] reported a photoswitchable pore blocker for the glutamate delta2 (GluD2) receptor. Glutamate delta receptors belong to the iGluR family due to their sequence and structure similarity to AMPA, NMDA and kainate receptors.…”

“…However, they are considered to be orphan receptors because their LBD does not bind glutamate; instead, pore opening is regulated indirectly by metabotropic G protein-coupled glutamate receptors. Hence, Lemoine and coworkers [ 122 ] devised a PTL based on a known pore blocker (pentamidine), instead of an agonist binding to the LBD. MAGu ( 21 ) is composed of maleimide, azobenzene and a guanidinium head group that mimics the positively charged groups of pentamidine.…”

“…However, differently to pentamidine, the blockade by MAGu was not dependent on membrane voltage, suggesting that the two molecules may have different binding sites. This prediction was tested by performing covalent docking and ion pore calculations [ 122 ]. The positively charged group of MAGu does not seem to reach the inside of the pore, in line with the different blocking properties of MAGu and pentamidine observed experimentally.…”

Tonic GluD1 channel current is independent of G protein activity in the dorsal raphe nucleus

“…Nonetheless, we and others have demonstrated that GluD1 R and GluD2 R channels carry ionic current upon activation of Gα q protein-coupled receptors (GqPCRs), either metabotropic glutamate (mGlu R , Ady et al, 2013; Benamer et al, 2018; Dadak et al, 2017) or α1-adrenergic receptors (Gantz et al, 2020), through a process that involves intact G protein signaling. Intriguingly, in cell lines and brain slices, GluD1 R and GluD2 R channels are open in the presumed absence of agonists and carry tonic cation current (Gantz et al, 2020; Lemoine et al, 2020). The origin of the tonic GluD R current is unknown.…”