A family of photoswitchable NMDA receptors
- University of California, Berkeley, United States
- Université de Nice Sophia Antipolis, France
- University of Munich, Germany
- Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 3 supplements
Photo-agonism of NMDA receptors in HEK293 cells and hippocampal neurons.
(a) MAG photoswitches showing chemical structure and cartoon depiction. MAG0 and MAG1 differ in length (brackets, n = 0, 1) (Gorostiza et al., 2007), whereas L-MAG and D-MAG (Levitz et al., 2013) …
Figure 1—figure supplement 1
Screen of GluN2A cysteine positions and MAG variants.
(a) Amino acids near the glutamate binding site of GluN2A were individually replaced with cysteine by site-directed mutagenesis, co-transfected with wild-type GluN1a into HEK293 cells, and tested …
Figure 1—figure supplement 2
Pharmacological characterization of GluN2A(V713C), GluN2A(G712C), GluN2B(V714C) and GluN1a(E406C).
Wild-type (wt) and engineered GluNRs were expressed in Xenopus oocytes and ligand-induced currents were recorded by two-electrode voltage-clamp (Vm = −60 mV, 0 Mg2+). (a) GluN1a-wt and GluN2A (wt, …
Figure 1—figure supplement 3
Lack of perturbation of neurons by MAG or LiGluN2A(G712C).
(a) MAG-exposure does not cause cell death or damage membrane integrity of cultured hippocampal neurons. Representative images of neurons (15 DIV) from wt animals following incubation for 45 min …
Figure 2 with 2 supplements
Rapid development of light-agonized LiGluN2B subunit, based on LiGluN2A.
(a) Partial sequence alignment (left) and overlaid crystal structures (ribbon) of the LBDs of GluN2A (blue, PDB-2A5S (Furukawa et al., 2005)) and GluN2B (grey, PDB- 4PE5 [Karakas and Furukawa, 2014])…
Figure 2—figure supplement 1
Photo-activation of LiGluN2A and -2B drives action potential firing in hippocampal neurons.
(a-b) Representative traces (current clamp) of neurons transfected with LiGluN2A(V713C) (a, black trace) or LiGluN2B(V714C) (b, grey); labeled with L-MAG1 and illuminated with 380 nm (violet bars) …
Figure 2—figure supplement 2
Summary of nonparametric statistics for Figure 2.
Results from Normality and nonparametric Mann-Whitney Rank Sum Tests are displayed for results shown in Figure 2d–f.
Figure 3 with 1 supplement
Photo-antagonism of NMDA receptors in hippocampal neurons.
(a) Photo-antagonism with L-MAG0 attached to LiGluN2A(G712C), where the cis-configuration is thought to place the glutamate end of MAG near the binding pocket, where it impedes LBD closure or entry …
Figure 3—figure supplement 1
Moderate correlation between total current size and photo-current.
(a) Representative traces of photo-antagonism (left) and photo-agonism (right), displaying the relative sizes of the total NMDA-induced current and the photo-responses (inhibition/activation), …
Figure 4 with 2 supplements
Photo-inhibition of neuronal activity with LiGluN1a and LiGluN2A.
(a) Representative trace showing a long recording (current clamp) of a neuron (from wt rats) transfected with LiGluN1a(E406C) and LiGluN2A(G712C), labeled with L-MAG0, and illuminated with 380 nm …
Figure 4—figure supplement 1
Photo-antagonism of NMDA receptors in hippocampal neurons inhibits the development of EPSCs.
(a) Representative trace (voltage clamp, −60 mV) of a hippocampal neuron (from wt rats) transfected with LiGluN1a(E406C) and LiGluN2A(G712C), labeled with L-MAG0. During green light illumination …
Figure 4—figure supplement 2
Summary of nonparametric statistics for Figure 4.
Results from Normality and nonparametric Mann-Whitney Rank Sum Tests are displayed for results shown in Figure 4c–e and for Figure 4—figure supplement 1b–d.
Figure 5 with 1 supplement
Photo-block of synaptic transmission by LiGluNs in hippocampal autapses.
(a) (left) Representative images of hippocampal neurons (from wt rats) grown in low density forming autapses. (right) Schematics of hippocampal autapse used to measure the effect on the NMDA …
Figure 5—figure supplement 1
Summary of nonparametric and parametric statistics for Figure 5.
(a) Results from Normality tests and nonparametric Kruskal-Wallis One Way Analysis of Variance (ANOVA on ranks) are displayed for panels shown in Figure 5b and One Way ANOVA for Figure 5f. (b) No …
Figure 6
LTP induction blocked by photo-antagonism of LiGluN1a(E406C) and LiGluN2A(G712C) in organotypic hippocampal slice from GluN2A-knockout neonate mice.
(a) Schematic of the hippocampus with stimulating electrode on Schaffer collaterals CA3 pyramidal axons that innervate pyramidal neurons of CA1 and recording pipette on a transfected CA1 neuron from …
Figure 7
GluN2A(V713C) photo-agonism triggers calcium increase and expansion in single dendritic spines of organotypic hippocampal slice from GluN2A-knockout neonate mice.
(a) Schematic of photo-agonism with L-MAG1 conjugated to the LiGluN2A(V713C) subunit. (b, c) Single-spine calcium rise induced by photo-agonism. (b) CA1 neurons co-expressing LiGluN2A(V713C) fused …
Figure 8 with 1 supplement
Photo-antagonism prevents glutamate-induced expansion in single dendritic spines of organotypic hippocampal slice from GluN2A-knockout neonate mice.
(a) Schematic of a photo-antagonized NMDA receptors containing LiGluN1a(E406C) and LiGluN2A(G712C) conjugated to L-MAG0. (b) CA1 pyramidal neuron co-expressing LiGluN1a(E406C), LiGluN2A(G712C) and …
Figure 8—figure supplement 1
Single spine spatial precision of photocontrol in organotypic hippocampal slice from GluN2A-knockout neonate mice.
(a) Photo-bleaching indicates spatial precision of "on-spine" photo-stimulation. High intensity illumination at 405 nm (magenta arrow) rastered over a single 1 µm diameter spine for 1 s (white …
Figure 9 with 1 supplement
GluN2A(G712C) photo-antagonism disrupts refinement of retinal ganglion cell axon arbors in larval zebrafish in vivo.
(a) Cartoons depicting GluN2A(G712C) photo-antagonism (top left), development of retinal ganglion cell projection (top, right) and timeline of the photo-antagonism assay (bottom). (b) (Left) Dorsal …
Figure 9—figure supplement 1
MAG treatment of zebrafish larvae in the absence of LiGluN2A expression does not affect behavior.
Wildtype larvae were treated at 5 dpf with either 150 µM MAG0 in 0.3% DMSO (n = 48) or 0.3% DMSO alone (n = 48) for 40 min, rinsed thoroughly in fresh fish water, and allowed to develop as two …
