Extended Data Fig. 3: Structural, biochemical and electrophysiological analysis of N1a-N2D receptors.
From: Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits
a,b, Conformation comparison of NTDs (a) and protomers (b) between Gly-Glu bound N1-N2D and zinc-bound N1-N2A (PDB:6MMK, ref. 22) di-receptors. Lines, angles and arrows are illustrated as in Fig. 2 legend. c-e, Cartoon representation of the tetrameric interface formed by two N2D-NTDs (c) and two N1-LBDs (d) in Gly-Glu bound N1-N2D receptor, and formed by two N2A-NTDs (d) in Gly-Glu bound N1-N2A receptor (PDB:6MMP, ref. 22), with residues shown in sticks and Cα-Cα distances indicated. f-h, Western blotting analysis on WT and cysteine-substituted N1-N2A (f) and N1-N2D (g, h) receptors (repeated three times). Bands of N1 and N2 momomers, N1-N2 and N2-N2 dimers are indicated. i-j, Representative recording traces and relative MK-801 inhibition on-rate kinetics constants (τon, monoexponential fits) of WT N1-N2D (1.00 ± 0.07, n = 15), N1-N2DS238C(1.01 ± 0.07, n = 12, in i), N1-N2DL822C (0.62 ± 0.08, n = 6, in j), N1E698C-N2D (0.10 ± 0.01, n = 5, in j) and N1E698C-N2DL822C (0.09 ± 0.01, n = 5, in j) receptors. k,l, DTT induced current amplitude changes on WT and mutant receptors. Relative currents (after and before DTT treatment) values, from left to right, are 0.91 ± 0.08 (n = 4), 0.73 ± 0.03 (n = 4), 0.38 ± 0.08 (n = 3) and 0.57 ± 0.13 (n = 5) in k, and 3.56 ± 0.80 (n = 9), 0.65 ± 0.07 (n = 4) and 1.33 ± 0.10 (n = 4) in l. N1* implies the mutant N1C744A, C798A subunit. m, Dose-response curves (fitted by Hill equation) of glycine and glutamate on N1-N2D (Gly EC50 of 0.08 ± 0.01 μM, nH = 1.2 and Glu EC50 of 0.42 ± 0.04 μM, nH = 1.8) and N1E698C-N2D (Gly EC50 of 17.49 ± 0.26 μM, nH = 2 and Glu EC50 of 1.66 ± 0.05 μM, nH = 1.7) receptors. n = 4 oocytes for each group. All data are shown with mean ± SD. For the statistical analysis, P values are determined by two-tailed unpaired Student’s t-test for (i) and by one-way ANOVA followed by the Tukey’s multiple comparison test for (j-l).
