The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The Kd values for binding of [14C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [3H]Ifenprodil bound to NR1‐R (Kd, 0.18 μM) and NR2B‐R (Kd, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These Kd values were confirmed by circular dichroism measurements. The Kd values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.

The transmembrane and pore-forming regions of N-methyl-Daspartate receptors containing the NR1 and NR2B subunits were studied by measuring the effects of various NR1 and NR2B mutants on stimulation and block by spermine. Block by spermine was predominantly affected by mutations in the M3 segment of NR1 and especially in the M1 and M3 segments of NR2B. These regions are in the outer vestibule of the channel pore and may contribute to a spermine binding site. Mutations in different regions-predominantly the M3 segment and M2 loop of NR1 and the M3 segment of NR2B-influenced spermine stimulation, a surprising finding because spermine stimulation is thought to involve a spermine binding site in the distal, extracellular regulatory domain. However, some of these mutations also influence sensitivity to ifenprodil and protons, and changes in spermine sensitivity may be secondary to changes in proton sensitivity. The results are consistent with the proposal that the relative positions of the M1 and M3 transmembrane segments and M2 loops are staggered or asymmetric in NR1 and NR2 subunits, and with the idea that stimulation and block by spermine involve separate binding sites and distinct mechanisms, although some residues in the receptor subunits can affect both stimulation and block.

Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N-methyl-D-aspartate receptorGluN1 and GluN2B subunits was carried out after measuring spermine stimulation and ifenprodil inhibition at receptors containing GluN1 and GluN2B R domain mutants. Models were constructed based on the published crystal structure of the GluN1 and GluN2B R domains, which form a heterodimer (Nature 475: 249 -253, 2011). The experimental results and modeling suggest that a binding site for spermine was formed by the residues near the cleft between the R1 and R2 lobes of the GluN1 R domain (GluN1R) together with residues on the surface of the R2 (C-terminal side) lobe of the GluN2B R domain (GluN2BR). The ifenprodil binding site included residues on the surface of the R1 lobe (N-terminal side) of GluN1R together with residues near the cleft between the R1 and R2 lobes of GluN2BR. It was confirmed using a Western blot analysis that GluN1R and GluN2BR formed a heterodimer. Models of spermine and ifenprodil binding to the heterodimer were constructed. The modeling suggests that an open space between the two R1 lobes of GluN1R and GluN2BR is promoted through spermine binding and that the R1 lobes of GluN1R and GluN2BR approach each other through ifenprodil binding-an effect opposite to that seen with the binding of spermine.