bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.093419; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Probing the ionotropic activity of the orphan glutamate delta 2 receptor with 2 genetically-engineered photopharmacology. 3 4 5 6 Damien Lemoine1, Sarah Mondoloni1, Jérôme Tange1, Bertrand Lambolez1, Philippe 7 Faure1, Antoine Taly2,3*, Ludovic Tricoire1* and Alexandre Mourot1*. 8 9 10 1 Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), CNRS, 11 INSERM, Sorbonne Université, Paris, France 12 2 CNRS, Université de Paris, UPR 9080, Laboratoire de Biochimie Théorique, UPR 13 9080, 13 rue Pierre et Marie Curie, F-75005, Paris, France 14 3 Institut de Biologie Physico-Chimique-Fondation Edmond de Rothschild, PSL 15 Research University, Paris, France 16 * Equal contribution 17 18 19 Correspondence to: [email protected] ; [email protected] 20 21 22 23 Keywords: optogenetics, photopharmacology, Glutamate receptors, tethered ligands, 24 ion channels, azobenzene 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.093419; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 Abstract 27 Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, 28 yet whether they actually form functional and physiologically-relevant ion channels in 29 neurons remains a debated question. Here we used a chemo-genetic approach to 30 engineer specific and photo-reversible pharmacology in the orphan GluD2 receptor. 31 We incorporated a cysteine mutation in the cavity located above the putative ion 32 channel pore, for site-specific conjugation with a photoswitchable ligand. We first 33 showed that, in the constitutively-open GluD2 Lurcher mutant, current could be rapidly 34 and reversibly decreased with light. We then transposed the cysteine mutation to the 35 native receptor, to demonstrate with absolute pharmacological specificity that 36 metabotropic glutamate receptor signaling opens the GluD2 ion channel in 37 heterologous expression system. Our results assess the functional relevance of GluD2 38 ion channel and introduce an optogenetic tool that will provide a novel and powerful 39 means for probing GluD2 ionotropic contribution to neuronal physiology. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.093419; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 40 Glutamate delta (GluD1 and GluD2) receptors are considered orphan because, 41 while having a strong sequence homology with the other ionotropic glutamate 42 receptors (AMPA, NMDA and Kainate), they are not activated by glutamate1,2. GluD 43 receptors are both widely expressed throughout the brain, GluD1 predominating in the 44 forebrain, and GluD2 being highly enriched in cerebellar Purkinje neurons3,4. Both 45 GluD1 and GluD2 play a role in the formation, stabilization, function and plasticity of 46 synapses4-7. Likewise, deletion of GluD1 or GluD2 genes in mouse results in marked 47 behavioral alterations8,9, and mutations in human GluD1 and GluD2 genes have been 48 associated with neurodevelopmental and psychiatric diseases10,11, attesting to their 49 functional importance in brain circuits. Nevertheless, due to the absence of 50 pharmacology, a detailed understanding of how GluD1/2 regulate specific neural 51 circuits, and notably whether their ionotropic activity is involved, is lacking. 52 53 Although GluD1 and GluD2 exhibit a domain similar to the ligand binding domain (LBD) 54 of other iGluRs12, no ligand has been found that directly triggers the opening of the 55 pore. Yet, several observations indicate that the ion channel of GluD receptors may be 56 functional. First, crystallization studies show that the LBD of GluD2 binds D-serine and 57 glycine, and that these ligands induce “agonist-like” structural rearrangements in the 58 LBD, even though they fail to evoke currents at wild-type (WT) GluD receptors 59 expressed in heterologous expression systems13. Second, a point mutation (A654T) in 60 GluD2 that causes the degeneration of cerebellar Purkinje neurons in Lurcher (Lc) 61 mice confers constitutive ion flow14,15. Current through GluD2Lc receptors is inhibited 62 by pentamidine and 1-Naphthyl acetyl spermine (NASPM)16,17, pore blockers of NMDA 63 and AMPA receptors, respectively. Furthermore, D-serine and glycine reduce the 64 spontaneous currents of GluD2Lc, suggesting a coupling between the LBD and the 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.093419; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 65 channel13,18. Third, receptor chimeras containing the LBD of AMPA receptors and the 66 membrane domain of GluD receptors show glutamate-induced currents19. Fourth, the 67 GluD1/2 receptor channel can be opened following activation of type I metabotropic 68 glutamate receptors (mGlu1/5), and these currents are almost completely blocked by 69 NASPM and reduced by D-serine20-22. Finally, the slow excitatory postsynaptic currents 70 observed in midbrain dopaminergic, dorsal raphe, and cerebellar Purkinje neurons, are 71 abolished upon gene inactivation or expression of dominant-negative pore mutants of 72 GluD1/220,22,23. All these findings indicate that GluD receptors likely possess a 73 functional ion channel pore. Yet, a direct evidence for ionotropic activity of GluD in 74 neuronal setting is lacking, due to the inability to specifically and acutely block GluD 75 conductance. 76 77 To fill this gap, we bestowed light-sensitivity to the GluD ion channel pore using 78 an optogenetic pharmacology approach24. We incorporated a cysteine point mutation 79 at the surface of GluD2, right above the hypothetical channel lumen, onto which can 80 be anchored a photoswitchable tethered ligand (PTL). Light is then used to modify the 81 geometry of the PTL, thereby presenting/removing the ligand to/from the channel, 82 resulting in optical control of ionotropic activity. Here we demonstrate rapid and 83 reversible, optical control of ion current through a cysteine-substituted GluD2 receptor. 84 This novel tool, called light-controllable GluD2 (LiGluD2), allows rapid, reversible and 85 pharmacologically-specific control of GluD2, and may help provide a mechanistic 86 understanding of how this receptor contributes to brain circuits and behaviors. 87 88 Our approach to probing the functionality of the ion channel in GluD is to install 89 a photo-isomerizable pore blocker at the extracellular entrance to the channel lumen 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.093419; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 90 (Figure 1A). The tethered ligand is site-specifically attached to a cysteine-substituted 91 residue. In darkness or under green light, the PTL adopts an elongated shape and 92 reaches the lumen, resulting in ion channel blockade, while under violet light, it 93 switches to a twisted, shorter configuration, relieving blockade. Our design of the PTL 94 was based on the chemical structure of pentamidine (Figure 1B), a pore blocker that 95 efficiently blocks current through GluD2Lc receptors17. The PTL, called MAGu, contains 96 a thiol-reactive maleimide (M) moiety, a central photo-isomerizable azobenzene (A) 97 chromophore, and a guanidinium (Gu) head group that resembles the amidinium 98 groups of pentamidine (Figure 1C). MAGu was selected notably because its synthesis 99 route has been described (referred to as PAG1c in the original article25). In aqueous 100 solution, MAGu could be converted to its cis form using 380 nm light, and converted 101 back to trans either slowly in darkness (t1/2 ~ 20 min) or rapidly upon illumination with 102 525 nm light (Supp. Fig.1A-B), in agreement with previous reports26. To find the best 103 attachment site for MAGu on GluD, we developed a homology model of the GluD2 104 receptor, based on the structure of the recently crystallized GluA2 receptor27 (see 105 methods). Using this model, we selected a series of 15 residues, located on the peptide 106 that links the LBD to the third transmembrane domain (TM3) that lines the channel 107 lumen, for mutation to cysteine (Figure 1D-E). 108 109 Since no known ligand directly gates the ion channel of GluD2, we used a Lc 110 mutant, L654T, which displays a constitutively open channel14,15, for screening the 15 111 single-cysteine mutations. Accordingly, we found that heterologous expression of 112 GluD2-L654T, but not of the wild-type (WT) protein, in HEK cells produces large 113 currents that reverse at membrane potential close to 0 mV and are reduced by 114 externally-applied pentamidine (Figure 2A). Subtracted Lc current showed clear 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.093419; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 115 rectification at positive potentials, as reported with the blockade by NASP another GluD 116 blocker16. Therefore, the L654T Lc mutant was subsequently used as a screening 117 platform to find the best attachment site for MAGu on GluD2. Each of the 15 residues 118 identified in Fig. 1D were mutated individually to cysteine on the L654T background, 119 and tested using patch-clamp electrophysiology. Cells were treated with MAGu (20 120 µM, 20 min) and Lc currents were measured in voltage-clamp mode (-60 mV) under 121 different illumination conditions to toggle MAGu between its cis and trans states.