Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with genetically-engineered photopharmacology
- Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), CNRS, INSERM, Sorbonne Université, France
- CNRS, Université de Paris, UPR 9080, Laboratoire de Biochimie Théorique, France
- Institut de Biologie Physico-Chimique-Fondation Edmond de Rothschild, PSL Research University, France
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 …
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Figure 2—source data 1
Related to Figure 2A, B and C.
- https://cdn.elifesciences.org/articles/59026/elife-59026-fig2-data1-v2.xlsx
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 …
Figure 3
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 …
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Figure 3—source data 1
Related to Figure 3A, C and D.
- https://cdn.elifesciences.org/articles/59026/elife-59026-fig3-data1-v2.xlsx
Figure 4
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 …
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Figure 4—source data 1
Related to Figure 4A and B.
- https://cdn.elifesciences.org/articles/59026/elife-59026-fig4-data1-v2.xlsx
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 …
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Figure 5—source data 1
Related to Figure 5C and D.
- https://cdn.elifesciences.org/articles/59026/elife-59026-fig5-data1-v2.xlsx
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 | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | HEK tsa201 | Sigma-Aldrich #96121229 | RRID:CVCL_2737 | |
| Chemical compound, drug | MAGu | 10.1016/j.neuron.2015.10.026 | Originally named PAG-1c. Custom-synthetized by Enamine, Ukraine | |
| Chemical compound, drug | DMSO | Sigma-Aldrich | D2650 | |
| Chemical compound, drug | Pentamidine | Sigma-Aldrich | 1504900 | |
| Chemical compound, drug | N-methyl-d-glucamine (NMDG) | Sigma-Aldrich | M2004 | |
| Chemical compound, drug | (R,S)−3,5-DHPG | Hello-bio | HB0026 | |
| Chemical compound, drug | CNQX | Hello-bio | HB0205 | |
| Chemical compound, drug | D-APV | Hello-bio | HB0225 | |
| Chemical compound, drug | SR 95531 (Gabazine) | Hello-bio | HB0901 | |
| Chemical compound, drug | CGP 55845 | Hello-bio | HB0960 | |
| Gene (Mus musculus) | Grid2 (glutamate receptor, ionotropic, delta 2) | Genbank | GeneID: 14804 | |
| Gene (Rattus norvegicus) | Grm1 (glutamate receptor, metabotropic 1) | Genbank | Gene ID: 24414 | |
| Transfected construct (Mus musculus) | pcDNA3-GluD2 | https://doi.org/10.1002/embr.201337371 | NM_008167.3 | |
| Transfected construct (Mus musculus) | pRK5-mGlu1b | Laurent Prezeau (IGF, Montpellier, France). | NM_001114330.1 | |
| Software, algorithm | R Project for Statistical Computing | http://www.r-project.org/ | RRID:SCR_001905 | |
| Software, algorithm | Modeller 9.19 | https://salilab.org/modeller/9.19/release.html | RRID:SCR_008395 | |
| Software, algorithm | Smina | https://sourceforge.net/projects/smina/ |
