Identification of a steroid modulatory domain (SMD1) in NR2B. (a Left) A schematic representation of the WT NR2B, NR2D, and the NR2B/NR2D chimeras is displayed. Vertical bars correspond to the four hydrophobic domains. The contribution of NR2B and NR2D to chimeras is depicted in black and white, respectively. The scales at the top indicate the residue numbers in the WT subunits at junctions. Vertical bars represent the four hydrophobic membrane domains. (a Right) The percentage increase in the NMDA/glycine response (elicited by 300 μM NMDA and 50 μM glycine in oocytes expressing NR1-1a and NR2 subunits) in the presence of 100 μM PS is indicated. Error bars are SEMs. Numbers adjacent to the error bars indicate the number of oocytes used in the study. (b) Concentration–response curves of PS modulation for receptors containing NR2B (○), NR2D (▵), χ4(□), and χ5(⋄), were determined in the presence of saturating concentrations of NMDA (300 μM) and glycine (50 μM). The EC₅₀ (NMDA) for NR1-1a/χ4 and NR1-1a/NR2B are both 22 ± 1 μM, and EC₅₀ (glycine) is 0.3 ± 0.02 μM and 0.1 ± 0.02 μM, respectively. (c) The topological representation of the NR2B subunit and the location of the identified segment (colored in red) are depicted. Membrane domains are denoted as M1–M4. The amino terminus (N) is located on the extracellular side and the carboxyl terminus (C) on the intracellular side of the plasma membrane.

SMD1 is essential for positive and negative modulation by spermine and protons but not for negative modulation by 3α5βS. (a) Percentage change of NMDA/glycine current elicited by application of 300 μM NMDA plus 50 μM glycine in the presence of 100 μM PS, 100 μM3α5βS, or 100 μM spermine. (b) Proton inhibition concentration–response relationship for NMDA/glycine responses of receptors containing NR1-1a and NR2B (•, n = 10; pK_a = 7.4 ± 0.1) or χ4(○, n = 10; pK_a = 6.8 ± 0.2); P < 0.01. Responses are expressed as a percentage of the value at pH 9. (c) Spermine potentiation concentration–response curves for receptors containing NR1-1a and NR2B at pH 7.5 (•, n = 7), χ4atpH7.5 (○, n = 8), or χ4atpH6.6 (□, n = 8). Vh =–70 mV except in spermine modulation experiments where Vh = –20 mV. Error bars are SEMs. Numbers adjacent to the error bars are the number of oocytes used in this study.

Reducing proton sensitivity through mutation of the NR1-1a subunit at E342 does not reduce PS modulation. (a) NR1-1a(E342Q)/NR2B shows a WT response to PS. Concentration–response curves for PS positive modulation of NMDA receptor activity for NR1-1a/NR2B (black), NR1-1a/χ4 (purple), and NR1-1a(E342Q)/NR2B (red). Percentage increases in NMDA/glycine-elicited currents by PS are plotted as a function of the concentration of PS (n = 12–18). (b) NR1-1a/χ4 shows a WT response to ifenprodil. Concentration–response curves for ifenprodil inhibition of NMDA receptor activity for NR1-1a/NR2B (black), NR1-1a/χ4 (purple), and NR1-1a(E342Q)/NR2B (red). All points are normalized to NMDA/glycine currents in the absence of ifenprodil (n = 10–15). Oocytes were clamped at Vh = –40 mV to eliminate the voltage-dependent blockade by ifenprodil.

Molecular modeling of a dimer comprising the S1/S2 domains of NR2B and NR1-1a reveals a potential binding pocket for PS. (a) The 3D model is depicted in a ribbon configuration with α helices colored in blue and β sheets colored in yellow. PS is docked at the interface between two subunits. Detailed views of the potential binding pocket for PS on NR2B (b) and NR1-1a (c) are illustrated. NR1-1a or NR2B is removed from the model to show the hydrophobic pocket on NR2B (b) or NR1-1a (c), respectively. The receptor surface is colored according to a hydrophobicity scale. Hydrophobic residues are colored in red and charged residues in blue. PS is depicted in a stick configuration and colored by the atom type with hydrogen in white, carbon in green, oxygen in red, and sulfur in yellow.

Multiple residues in SMD1 participate in modulation of NMDA receptor function by PS. (a) Multiple sequence alignment of the identified segment in NR2A, NR2B, NR2C, and NR2D of the NMDA receptor and GluR2 of the AMPA receptor. Locations of J/K helices and the M4 domain are indicated by rectangular bars. Residues of GluR2 at RNA editing (▾) and the alternative splicing (▴) sites are marked underneath the GluR2 sequence. Residues subjected to site-directed mutagenesis are indicated by black stars (★). (b) The residues of NR2B (★) were replaced by the corresponding residues of NR2D. Responses of receptors containing the WT NR2B, χ4 and the single mutants to PS are presented as percentage increases in NMDA/glycine currents in the presence of 100 μM PS. All NR2 subunits were coexpressed with NR1-1a. Error bars are SEMs. Numbers adjacent to the error bars are the number of oocytes used in this study. †, Statistically significant reduction in PS potentiation (P < 0.05). Red star, position of Q812 that reduces PS potentiation by 45 ± 5%. N.A., No currents were recorded from oocytes injected with mRNA of NR1-1a and T835V NR2B mutant.

The proton sensor does not control steroid modulation. Percentage change of NMDA/glycine currents elicited by 300 μM NMDA + 50 μM glycine to NR1-1a or NR1-1b with NR2B (Left) or χ4(Right) in the presence of 100 μM PS (a;Vh = –70 mV) or 100 μM spermine (b;Vh = –20 mV) at pH 7.5 or 6.6.

Proposed model for SMD1-mediated allosteric modulation of agonist-induced gating of NR2B containing NMDA receptors. Diagram shows convergence of independent pathways for positive allosteric modulation by PS (+) and negative allosteric modulation by H⁺ (–) of neurotransmitter (glutamate and glycine)-induced gating of the NMDA receptor through SMD1. SMD1 spans the J/K helices and M4 regions of NR2B and is located near the glutamate-binding site.