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.

26Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, 27 yet whether they actually form functional and physiologically-relevant ion channels in 28 neurons remains a debated question. Here we used a chemo-genetic approach to 29 engineer specific and photo-reversible pharmacology in the orphan GluD2 receptor. 30We incorporated a cysteine mutation in the cavity located above the putative ion 31 channel pore, for site-specific conjugation with a photoswitchable ligand. We first 32 showed that, in the constitutively-open GluD2 Lurcher mutant, current could be rapidly 33 and reversibly decreased with light. We then transposed the cysteine mutation to the 34 native receptor, to demonstrate with absolute pharmacological specificity that 35 metabotropic glutamate receptor signaling opens the GluD2 ion channel in 36 heterologous expression system. Our results assess the functional relevance of GluD2 37 ion channel and introduce an optogenetic tool that will provide a novel and powerful 38 means for probing GluD2 ionotropic contribution to neuronal physiology. 393 Glutamate delta (GluD1 and GluD2) receptors are considered orphan because, 40 while having a strong sequence homology with the other ionotropic glutamate 41 receptors (AMPA, NMDA and Kainate), they are not activated by glutamate 1,2 . GluD 42 receptors are both widely expressed throughout the brain, GluD1 predominating in the 43 forebrain, and GluD2 being highly enriched in cerebellar Purkinje neurons 3,4 . Both 44GluD1 and GluD2 play a role in the formation, stabilization, function and plasticity of 45 synapses 4-7 . Likewise, deletion of GluD1 or GluD2 genes in mouse results in marked 46 behavioral alterations 8,9 , and mutations in human GluD1 and GluD2 genes have been 47 associated with neurodevelopmental and psychiatric diseases 10,11 , attesting to their 48 functional importance in brain circuits. Nevertheless, due to the absence of 49 pharmacology, a detailed understanding of how GluD1/2 regulate specific neural 50 circuits, and notably whether their ionotropic activity is involved, is lacking. 51 52 Although GluD1 and GluD2 exhibit a domain similar to the ligand binding domain (LBD) 53 of other iGluRs 12 , no ligand has been found that directly triggers the opening of the 54 pore. Yet, several observations indicate that the ion channel of GluD receptors may be 55 functional. First, crystallization studies show that the LBD of GluD2 binds D-serine and 56 glycine, and that these ligands induce "agonist-like" structural rearrangements in the 57 LBD, even though they fail to evoke currents at wild-type (WT) GluD receptors 58 expressed in heterologous expression systems 13 . Second, a point mutation (A654T) in 59GluD2 that causes the degeneration of cerebellar Purkinje neurons in Lurcher (Lc) 60 mice confers constitutive ion flow 14,15 . Current through GluD2 Lc receptors is inhibited 61 by pentamidine and 1-Naphthyl acetyl spermine (NASPM) 16,17 , pore blockers of NMDA 62 and AMPA receptors, respectively. Furthermore, D-se...

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