Binding of spermine and ifenprodil to a purified, soluble regulatory domain of theN‐methyl‐d‐aspartate receptor

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Abstract
The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of theN‐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 inEscherichia coliand purified to near homogeneity. TheKdvalues 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). TheseKdvalues were confirmed by circular dichroism measurements. TheKdvalues 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.