Figures and data
ScRNA-seq and immunoprecipitation analysis of NMDAR subunits co-expression and co-assembly patterns in the brain.
a. Cartoon illustration depicting the brain regions analyzed in scRNA-seq and Immunoprecipitation. b. Co- expression analysis of Grin3a with other NMDAR subunits in the cortex, hippocampus, cerebellum, and basolateral amygdala. Colors represent the proportion of cells within each subclass expressing specific subunit combinations. c. Paired co-expression analysis of NMDAR subunits in cortex SST neuron, hippocampus CA1 neurons, cerebellum PLI neuron, and basolateral amygdala EX1 neurons, presented as heatmaps. Colors indicate the proportion of cells co-expressing indicated pairs of NMDAR subunits. d. Immunoprecipitation (IP) of GluN3A (N3A) with other NMDAR subunits. Total protein from the cortex and hippocampus was precipitated with anti-N3A and subsequently examined by Western blotting using antibodies against NMDAR subunits. Normal IgG (IgG) was used as a negative control. Flow-through (F.T.) fractions were examined to verify effective IP.
Purification, validation, and structural analysis of N1-N2A-N3A tri- NMDARs.
a. Topological representation and purification workflow of N1-N2A-N3A. A two-step affinity-tagged chromatographic process was used for the N1-N2A-N3A purification. Fractions containing N1-N2A or N1-N3A are also collected for further analysis. b. Coomassie blue staining and Western blot analysis of N1-N2A, N1-N3A, and N1-N2A-N3A receptors from the same preparation batch, post-digestion by Endo H and 3C protease. c. SEC traces of N1-N2A- N3A receptors. d. FSEC traces depicting the shifts in N1-N2A, N1-N3A, and N1-N2A-N3A receptors upon binding with specific fabs against N1 (clone 4F11), N2A (clone 28C), N3A (clone 1G4), or a combination of fabs against N1 and N2A, as indicated. e. Cryo-EM maps of N1-N2A- N3A bound with N1 and N2A Fabs, presented inside and top-down views. The four subunits (N1, N2A, N1, and N3A) are color-coded accordingly as gray, orange, blue, and gray. The N1- specific fab 4F11 is shown in dark gray, and the N2A-specific fab 28C is depicted in brown. f. The overall, NTD, and LBD structure of N1-N2A-N3A. g. The overall structure of NTD and LBD of N1-N2A and N1-N3A heterodimer. Superimposition of the NTD R1 lobes or LBD from N1- N2A and N1-N3A protomers of N1-2A-3A, or N1-N2A protomers from N1-N2A-N3A and 6MMP. Superimposition of the LBD from N1-N2A protomers of N1-N2A-N3A and crystal N1-N3A LBD structure(2RC7). h. Structural analysis of extracellular domains of each protomers. The center- of-mass (COM) of R1 and R2 of NTD, D1 and D2 of LBD of each protomer is indicated by open circles. The distance connected by COMs was measured.
Single-Channel Properties of N1-N2A, N1-N3A, and N1-N2A-N3A receptors
Single channel properties of proteoliposomes reconstituted with N1-N2A, N1-N3A, and N1-N2A-N3A receptors.
a. Cartoons illustration of proteoliposomes reconstituted with N1-N2A, N1-N3A, and N1-N2A-N3A receptors, respectively. b. Representative single-channel currents of reconstituted N1-N2A, N1- N3A, and N1-N2A-N3A receptors at a holding potential of -60 mV with indicated agonist stimulation. Highlighted regions are shown on an expanded scale. c. Histogram showing single-channel open probabilities of N1-N2A (n=5), N1-3A (n=8), N1-N2A-N3A with 100 uM Glu plus 3 uM Gly treatment (n=12) and N1-N2A-N3A with 100 uM Glu plus 100 uM Gly treatment (n=6). The overall open probability values, from left to right, are 0.46 ± 0.020, 0.052 ± 0.007, 0.047 ± 0.010, and 0.172 ± 0.014. d-g. Amplitude histograms of single-channel conductance for N1-N2A, N1-N3A, and N1-N2A-N3A, fitted with the Sum of two Gaussian distributions.
Probing the subunit geometry of N1-N2A-N3A and N1-N2B-N3A tri- receptors by click chemistry.
a. Introduction of AzF in and N1-N2A di-NMDARs and N1-N2A-N3A tri-NMDARs through co-transfection of plasmids encoding N1, N2A with TAG mutant, N3A, engineered tRNA synthetase (AzFRs), and suppressor tRNA (Yam) (Left). Cartoon and model indicating AzF introduction sites at α5 of N2A NTD in N1-N2A and N1-N2A-N3A receptor (Middle and Right). b. Introduction of AzF at the interface of two NMDAR subunits. Upon UV stimulation, AzF-incorporated subunits form a covalent bond with nearby residues, detectable by western. c. Representative western blots demonstrating antibody recognition of N1, N2A or N2B, and N3A from cells under different treatments as indicated above the panels. Crosslinked protein bands are indicated by receptor Icon. A non-specific band was detected by anti-GluN1 as indicated by asterisks.
Proving the existence and the subunit geometry of N1-N2A-N3A and N1-N2B-N3A by applying click chemistry in cultured hippocampus neurons.
a. b. Cultured neurons electroporated with bicistronic plasmids encoding engineered tRNA synthetase (AzFRs), suppressor tRNA (Yam), and N2A with K220AzF mutant. Electroporated neurons cultured with or without AzF. c. Representative western blots demonstrating antibody recognition of exogenously expressed GluN2A (Strep, c) or GluN2B (His, d), NTD of GluN2A or GluN2B, AzFRs (Flag), and MAP2 from cultured neurons electroporated with AzFRs-T2A-mRuby-Yam+hGluN2AK220AzF-GFP (c), AzFRs- T2A-mRuby-Yam+hGluN2BK221AzF-GFP (d) and cultured with or without AzF as indicated above the panels. Exaggerates GluN2A or GluN2B bands indicated by empty triangles, endogenous GluN2A or GluN2B by filled triangles. e, f. Representative blots for antibodies proved exogenous GluN2A (Strep, e) or GluN2B (His, f), NTD of GluN2A (e) or N2B (f), and GluN3A from Strep (e) or Ni-resin (f) purified examples extracted from the neurons electroporated with AzFRs-T2A-mRuby-4Yam+N2AK220AzF-GFP (e) or AzFRs-T2A-mRuby-Yam+hGluN2BK221AzF-GFP (f) and cultured with or without AzF as indicated above the panels. Crosslinked protein bands indicated by the arrowheads. g. Representative blots of GluN1 from 1% input for Strep purification.
Proving the Existence and Subunit Geometry of N1-N2A-N3A and N1-N2B-N3A through in vivo application of Click Chemistry.
a. Cartoon illustration depicting the in vivo application of click chemistry. b. Representative images of brain slices from in utero electroporated animal brains bred with or without AzF. The embryos underwent in utero electroporation with plasmids encoding the engineered tRNA synthetase (AzFRs), the suppressor tRNA (Yam), and GluN2A with the K220AzF mutant. After electroporating the DNA construct into a lateral ventricle, the embryos were gently inserted into the abdominal cavity. The pregnant mice were provided with AzF-containing water, and the brains were acquired at postnatal days 7-14. c-e. Total proteins from the cortical-hippocampal regions of AzF-incorporated animals or negative control animals were immunoprecipitated with anti-GluN2A (d) or anti-GluN2B (e). The immunoprecipitated were subsequently examined by Western blotting using anti- GluN2A, anti-GluN2B, and anti-GluN3A antibodies. The arrowhead, filled triangle, and open triangle indicate the bands corresponding to the crosslinked complex, endogenous GluN2A or GluN2B, and overexpressed GluN2A or GluN2B, respectively.
Structural comparation of N1-N2A-N3A with other NMDA receptors.
a Structural analysis of extracellular domains of N2A or N3A subunits. The COM of R1 and R2 of NTD, D1 and D2 of LBD of each protomer is indicated by open circles. The COMs angles were measured. b. LBD comparison of N1-N2A-N3A with N1-N2A-N2B and N1-N3A.
Violins plot showing the expression level of Grin1, Grin2a, Grin2b, and Grin3a from different cell types in mouse cortex and hippocampal formation.
Representative immunofluorescence image of brain slice (a) and cultured hippocampal neurons (b) stained of GluN3A (red) and MAP2 (green).
Generation and validation of NMDAR subunit-specific antibodies.
a. Cartton illustration outlining the antibodies generation process. Full-length GluN1-N2A receptors were used for the development of anti-GluN1 and anti-GluN2A antibodies, while full-length GluN1-N3A receptors were employed for the development of anti-GluN3A antibodies. b-d. Representative immunofluorescent staining of HEK GnTI- cells expressing GFP-tagged different subtypes of NMDARs using anti-GluN1 (clone No. 4F11, b), anti-GluN2A (clone No. 28C, c), and anti-GluN3A (clone No. 1G4, d) antibodies. e. Representative immunofluorescent staining of HEK GnTI- cells expression GFP-tagged wild-type GluN1-N2A, wild-type GluN1-N2D, and various chimeric GluN1-N2D receptors.
Overview of cryo-EM image processing and 3D reconstruction of Fabs bound GluN1-N2A-N3A receptor structures.
a, b, Flowchart of image processing and 3D reconstruction of Fabs bound GluN1-N2A-N3A receptors. The 2D class images show Fabs bound 2D views in different orientations. The 3D classes with well features were selected and combined through several rounds of 3D classification for final refinement. In panel b, the red and white arrowheads represent localization of the anti- GluN1 fab or anti-GluN2A fab, respectively. c, Fourier shell correlation (FSC) curve for the resolution estimation.
Sequence alignment of rat GluN1, GluN2A, and GluN3A.
Secondary structure elements of GluN1, GluN2A, and GluN3A are indicated in above sequence alignment. Invariant hand highly conserved amino acids are sharded yellow. Green GluN2A vs GluN3A, pink (GluN1 vs GluN3A), and violet (GluN1 vs GluN2A).
Channel currents from proteoliposomes reconstituted GluN1-N3A receptors with different concentration of Gly treatment .
Incorporation of the AzF into GluN2A through genetic code expansion.
a, b. Three plasmids encoding GluN1, and N2A with TAG mutant at the position of K220, and the engineered tRNA synthetase (AzFRs) and the suppressor tRNA (Yam) were transfected with HKE cells. Representative fluorescence images (a) and western blot (b) indicate the the incorporation of AzF into GluN2A. c. Three plasmids encoding N1, and N2A with TAG mutant at the position of K220, and the engineered tRNA synthetase (AzFRs) and the suppressor tRNA (Yam) were co-injected into Xenopus laevis oocytes. Injected oocytes were then cultured with or without AzF, and the current was recorded at 48 hr and 72 hr after injection. For each condition, more than 20 oocytes were tested. Only currents over 10 nA were plotted. d. Representative traces recorded on oocytes injected with GluN1-N2A WT and GluN1-N2A K220AzF and UV stimulation. e. Representative blot for antibodies against GluN1 and GluN2A from oocytes injected GluN1- 2A K220AzF with or without UV stimulation. f. Four plasmids encoding GluN1, GluN2A with TAG mutant at the position of K220, the engineered tRNA synthetase (AzFRs), and the suppressor tRNA (Yam), GluN2B were transfected with HEK cells. Transfected cells were cultured with AzF, and the AzF-incorporated GluN1-N2A-N2B receptors were purified with Strep resin and analyzed by western blot. Representative blots for antibodies proved GluN2A (Strep) and GluN2B (Flag) from cells with different treatments as indicated on the top of this panel.
Single-Channel Properties of N1-N3A receptors.
