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

  1. Damien Lemoine
  2. Sarah Mondoloni
  3. Jérome Tange
  4. Bertrand Lambolez
  5. Philippe Faure
  6. Antoine Taly
  7. Ludovic Tricoire  Is a corresponding author
  8. Alexandre Mourot  Is a corresponding author
  1. Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), CNRS, INSERM, Sorbonne Université, France
  2. CNRS, Université de Paris, UPR 9080, Laboratoire de Biochimie Théorique, France
  3. Institut de Biologie Physico-Chimique-Fondation Edmond de Rothschild, PSL Research University, France
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Optogenetic pharmacology strategy to probe the ionotropic activity of GluD receptors.

(A) GluD2 is genetically-modified to incorporate a cysteine residue (yellow) at the entrance to the pore, which serves as a handle for the covalent attachment of a synthetic, photoswitchable …

Figure 1—figure supplement 1
Photochemical properties of MAGu.

(A) UV-visible spectra of MAGu under 520 nm light (green, mostly trans) or under 390 nm light (pink, mostly cis). (B) Thermal relaxation of cis MAGu in aqueous solution in darkness. Absorbance at …

Figure 2 with 1 supplement
Screening of the fifteen single-cysteine mutants engineered on the Lc background.

(A) Left, representative current-voltage relationship for GluD2-A654T Lc, with (blue) and without (black) pentamidine (100 μM). The subtracted current (gray) shows clear inward rectification and …

Figure 2—figure supplement 1
Functional characterization of the cysteine mutants.

(A) Currents recorded at −60 mV for A654T and each of the fifteen cysteine mutants (n = 3–8 cells). (B) Pentamidine block for A654T and each of the fifteen cysteine mutants (n = 3–8 cells). Data are …

Photoregulation of GluD2-A654T-I677C labeled with MAGu.

(A) Representative current traces (Vm = −60 mV) recorded for A654T-I677C in darkness, then under 380 light (violet), upon application of pentamidine or NMDG (blue, 100 μM), and then under 535 nm …

Pharmacological action of MAGu at GluD2-A654T-I677C.

(A) Average current-versus-voltage relationship under 380 and 535 nm light (n = 21). S.E.M is shown is shade, and subtracted current in gray. (B) Left, representative dose-dependent blockade of the …

Figure 5 with 1 supplement
Photocontrol of GluD2-I677C (LiGluD2).

(A) Representative DHPG-induced current (measured as indicated by the red arrow) for a MAGu-treated (20 µM, 20 min) cell co-expressing mGlu1b and GluD2-I677C, under 380, 535 and 380 nm light. Note …

Figure 5—figure supplement 1
MAGu does not photosensitize native GluD and GluA currents in Purkinje cells.

(A) Top, DHPG (200 μM)-induced current in vehicle-treated cerebellar slices were of similar amplitude under 380 and 535 nm light (−800 ± 57 pA vs. −799 ± 57 pA, p>0.999, n = 10). Middle, DHPG (200 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Cell line (H. sapiens)HEK tsa201Sigma-Aldrich #96121229RRID:CVCL_2737
Chemical compound, drugMAGu10.1016/j.neuron.2015.10.026Originally named PAG-1c. Custom-synthetized by Enamine, Ukraine
Chemical compound, drugDMSOSigma-AldrichD2650
Chemical compound, drugPentamidineSigma-Aldrich1504900
Chemical compound, drugN-methyl-d-glucamine (NMDG)Sigma-AldrichM2004
Chemical compound, drug(R,S)−3,5-DHPGHello-bioHB0026
Chemical
compound, drug
CNQXHello-bioHB0205
Chemical compound, drugD-APVHello-bioHB0225
Chemical compound, drugSR 95531 (Gabazine)Hello-bioHB0901
Chemical compound, drugCGP 55845Hello-bioHB0960
Gene (Mus musculus)Grid2 (glutamate receptor, ionotropic, delta 2)GenbankGeneID: 14804
Gene (Rattus norvegicus)Grm1 (glutamate receptor, metabotropic 1)GenbankGene ID: 24414
Transfected construct (Mus musculus)pcDNA3-GluD2https://doi.org/10.1002/embr.201337371NM_008167.3
Transfected construct (Mus musculus)pRK5-mGlu1bLaurent Prezeau (IGF, Montpellier, France).NM_001114330.1
Software, algorithmR Project for Statistical Computinghttp://www.r-project.org/RRID:SCR_001905
Software, algorithmModeller 9.19https://salilab.org/modeller/9.19/release.htmlRRID:SCR_008395
Software, algorithmSminahttps://sourceforge.net/projects/smina/

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