Article Figures & Data
Figures
- Fig. 1.
Concentration–response data for AICP and DCS at human NMDA receptor subtypes. (A) Chemical structures of agonists at the GluN1 glycine binding site. (B) Representative recordings of concentration–response data for AICP in the presence of 100 µM glutamate at recombinant human GluN1/2A and GluN1/2C receptors. (C, D) Concentration–response data for (C) AICP and (D) DCS at human NMDA receptor subtypes in the presence of 100 µM glutamate. Data are mean ± S.D. from 4 to 15 oocytes and are normalized to the maximal response to glycine (100 µM) measured in the same recording. The EC50 values and relative agonist efficacy (i.e., maximal responses relative to glycine) are listed in Table 1.
- Fig. 2.
Relationship between the glutamate binding site and AICP activity. (A) Representative two-electrode voltage-clamp recordings of responses at rat GluN1/2A and GluN1/2C receptors to 100 µM glycine alone, 10 µM AICP alone, 100 µM glycine plus 10 µM AICP, or 100 µM glutamate plus 100 µM glycine. The mean responses normalized to control (glutamate plus glycine) ± S.E.M. from 6 to 8 oocytes are listed below the respective ligand applications. (B) Concentration–response data for AICP at rat NMDA receptor subtypes in the presence of either 30, 100, or 300 µM glutamate. Data are mean ± S.D. from 3 to 16 oocytes and are normalized to the maximal response to glycine (100 µM) plus the corresponding concentration of glutamate (30, 100, or 300 µM) measured in the same recording. The EC50 values and relative agonist efficacies (i.e., maximal responses relative to glycine) are listed in Supplemental Table 1.
- Fig. 3.
Activity of AICP and DCS at NMDA receptors with mutations in the glycine binding site. (A) Structure of glycine bound in the agonist binding site of the GluN1 subunit (PDB ID 5I57; Yi et al., 2016). Dashed lines indicate polar interactions between glycine (yellow carbon) and GluN1 residues (gray carbon). Mutated residues are indicated in bold text. (B–E) Concentration–response data for AICP (B and C) and DCS (D and E) at rat GluN1/2A and GluN1/2C receptors with mutations in the glycine binding site measured using two-electrode voltage-clamp recordings. The responses are normalized to the fitted maximal response. Data are mean ± S.D. from 3 to 29 oocytes, and the EC50 values are listed in Supplemental Table 2.
- Fig. 4.
Activity of AICP in the presence of high and low concentrations of glycine. (A) Representative two-electrode voltage-clamp recordings of responses at human GluN1/2B and GluN1/2C receptors to increasing concentrations of AICP in the continuous presence of 0.6 µM glycine plus 100 µM glutamate. White bars above the recordings indicate responses to 0.6 µM glycine in the absence of AICP. (B) Concentration–response data for AICP in the presence of 100 µM glutamate plus either 0.6 µM glycine (●) or 100 µM glycine (□) at human NMDA receptor subtypes (GluN1/2A–D). Data are normalized to the maximal response to glycine (100 µM) without AICP in the same recording. Data are mean ± S.D. from 4 to 10 oocytes. EC50 values and relative agonist efficacies (i.e., maximal responses relative to glycine) are listed in Supplemental Table 3.
- Fig. 5.
Relative agonist efficacy of AICP is controlled by residues in multiple receptor domains. (A) NMDA receptor structure (PDB ID 4PE5; Karakas and Furukawa, 2014) illustrating the subunit arrangement and the layered domain organization composed of the transmembrane domains (TMDs) and two extracellular layers formed by the agonist binding domains (ABDs) and the ATDs. Agonist binding sites for glycine in GluN1 and glutamate in GluN2 subunits as well as the known positions of homologous residues E790 in GluN2B and Q800 in GluN2C are highlighted. (B, C) Concentration–response data for (B) AICP and (C) DCS measured using two-electrode voltage-clamp recordings at rat wild-type and mutant GluN1/2B and GluN1/2C receptors. Data are mean ± S.D. from 8 to 18 oocytes. (D) Concentration–response data for AICP and DCS measured using two-electrode voltage-clamp recordings at rat GluN1/2A and GluN1/2C receptors and receptors with interchanged ATDs [GluN1/2A-(2C ATD) and GluN1/2C-(2A ATD)]. Data are mean ± S.D. from 6 to 29 oocytes. The EC50 values and relative agonist efficacies (i.e., maximal responses relative to glycine) are listed in Supplemental Table 4.
- Fig. 6.
Desensitization properties of NMDA receptors are not different for glycine and AICP responses. (A, B) Representative fast-application whole-cell patch-clamp of responses at rat GluN1/2B and GluN1/2C receptors to long exposures (2 seconds) with 100 µM glycine or 10 µM AICP in the continuous presence of 100 µM glutamate. The glycine (blue) and AICP (red) responses are shown on different time scales. (C) Scatterplot of rise times (10%–90%) of glycine and AICP responses from individual cells (D) Scatterplot of deactivation time constants (τdeactivation) of glycine and AICP responses from individual cells. (E) Scatterplot of the ratio between steady-state (SS) and peak amplitudes of glycine and AICP responses from individual cells. (F) Scatterplot of steady-state (SS) and peak amplitudes of AICP responses relative to the glycine response in the same cell. Mean ± S.D. is indicated in the scatterplots. N = 8 and N = 5 cells for GluN1/2B and GluN1/2C, respectively. *Statistically significantly different from the response to glycine at the same receptor subtype (P < 0.05, unpaired two-tailed t test). Values are listed in Table 2.
- Fig. 7.
AICP enhances responses from GluN1/2C receptors in the presence of physiologically relevant concentrations of glycine. (A) Representative two-electrode voltage-clamp recordings of responses at rat GluN1/2B and GluN1/2C receptors to increasing concentrations of AICP in the continuous presence of 10 µM glycine plus 300 µM glutamate. (B) Concentration–response data for AICP in the presence of 10 µM glycine plus 300 µM glutamate at rat GluN1/2A–D. Data are mean ± S.D. from six oocytes for each subtype. (C) Representative two-electrode voltage-clamp recordings of responses at rat GluN1/2B and GluN1/2C receptors to increasing concentrations of DCS in the continuous presence of 10 µM glycine plus 300 µM glutamate. (D) Concentration–response data for DCS in the presence of 10 µM glycine plus 300 µM glutamate at rat GluN1/2A–D. Data are mean ± S.D. from six to seven oocytes. (E, F) Representative two-electrode voltage-clamp recordings of responses at rat GluN1/2B and GluN1/2C receptors to (E) 1 µM glycine or (F) 10 µM glycine plus 300 µM glutamate with and without 1 µM AICP. Bar graphs of responses from rat GluN1/2A-D receptors to 1 µM AICP in the presence of either (E) 1 µM glycine or (F) 10 µM glycine plus 300 µM glutamate normalized to the response to 100 µM glycine plus 300 µM glutamate in the same recording. The responses are measured using two-electrode voltage-clamp electrophysiology and data are mean ± S.D. from 4 oocytes for each condition. *Statistically significantly different from the response in the absence of AICP at the same receptor subtype (P < 0.05, paired two-tailed t test).
Tables
- TABLE 1
Concentration–response data for glycine site agonists at NMDA receptor subtypes
Concentration–response data for glycine site agonists at recombinant human and rat NMDA receptor subtypes measured using two-electrode voltage-clamp electrophysiology in the presence of 100–300 µM glutamate.
Receptor Subtype Glycine Site Agonists Receptor Species EC50 (µM) pEC50 ± S.E.M. Rel. Imax (%) nH N GluN1/2A AICP Human 0.066 7.18 ± 0.02 100 ± 2 1.3 15 Rat 0.036 7.49 ± 0.05 92 ± 2 1.3 29 Glycine Human 1.2 5.91 ± 0.01 100 ± 1 1.7 6 d-Serine Human 1.8 5.75 ± 0.02 99 ± 1 1.8 5 DCS Human 22.0 4.65 ± 0.02 90 ± 2 1.3 6 Rat 18.0 4.75 ± 0.01 91 ± 1 1.6 13 GluN1/2B AICP Human 0.025 7.60 ± 0.19 10 ± 1 0.7 9 Rat 0.014 7.86 ± 0.03 9 ± 0 1.1 14 Glycine Human 0.58 6.24 ± 0.02 101 ± 1 2.2 6 d-Serine Human 0.98 6.01 ± 0.05 97 ± 2 1.2 5 DCS Human 7.3 5.14 ± 0.06 63 ± 2 1.1 4 Rat 8.3 5.08 ± 0.01 61 ± 0 1.4 12 GluN1/2C AICP Human 0.0017 8.77 ± 0.04 353 ± 6 0.9 16 Rat 0.0010 9.04 ± 0.03 339 ± 10 1.0 17 Glycine Human 0.33 6.48 ± 0.03 103 ± 1 1.7 5 d-Serine Human 0.47 6.33 ± 0.03 106 ± 1 1.6 5 DCS Human 4.4 5.36 ± 0.05 151 ± 5 1.3 4 Rat 2.8 5.55 ± 0.01 197 ± 2 1.5 18 GluN1/2D AICP Human 0.025 7.61 ± 0.03 27 ± 1 1.4 14 Rat 0.031 7.51 ± 0.02 26 ± 1 1.5 11 Glycine Human 0.14 6.84 ± 0.04 100 ± 2 1.6 5 d-Serine Human 0.35 6.46 ± 0.03 101 ± 1 2.0 5 DCS Human 3.1 5.51 ± 0.06 76 ± 3 1.5 6 Rat 3.2 5.50 ± 0.01 97 ± 0 1.6 6 N, number of oocytes; nH, Hill coefficient; (Rel.) Imax, fitted maximal response relative to the maximal response to glycine.
- TABLE 2
Time course of macroscopic NMDA receptor responses to glycine and AICP
Data are from whole-cell patch-clamp recordings of rat GluN1/2B and GluN1/2C receptors expressed in HEK-293 cells. Here, 100 µM glycine or 10 µM AICP were applied using rapid solution exchange in the continuous presence of 100 µM glutamate (see Materials and Methods). Steady-state (SS) and peak responses were normalized to the glycine response from the same cell. The deactivation time course was described using mono-exponential fits, and the time constant is listed (τdeactivation). N is the number of cells, and the values are mean ± S.E.M.
Response GluN1/2B GluN1/2C Glycine AICP Glycine AICP Rise time (10%–90%, ms) 16 ± 2 74 ± 12 6.2 ± 0.6 46 ± 2 SS/peak amplitude 89 ± 1 81 ± 3 100 ± 1 97 ± 1 Peak amplitude (% of glycine) 100 8.1 ± 1.1 100 388 ± 18 SS amplitude (% of glycine) 100 7.4 ± 1.0 100 376 ± 18 τdeactivatiom (s) 0.820 ± 0.033 22.6 ± 4.2 0.662 ± 0.021 84.3 ± 22.0 N 8 8 5 5
Data Supplement
- Supplemental Tables -
Supplemental Table 1. Influence of glutamate concentrations on AICP concentration-response data at rat
NMDA receptor subtypes.
Supplemental Table 2. Concentration-response data for AICP and DCS at wild type and mutant rat
GluN1/2A and GluN1/2C receptors.
Supplemental Table 3. Concentration-response data for AICP at human NMDA receptor subtypes in the
presence of glycine.
Supplemental Table 4. Concentration-response data for AICP and DCS at mutant and chimeric
GluN1/2A, GluN1/2B, and GluN1/2C receptors.
- Supplemental Tables -
