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

• Published in: Journal of neurochemistry 2008-12, Vol.107 (6), p.1566-1577
• Main Authors: Han, Xia, Tomitori, Hideyuki, Mizuno, Satomi, Higashi, Kyohei, Füll, Christine, Fukiwake, Tomohide, Terui, Yusuke, Leewanich, Pathama, Nishimura, Kazuhiro, Toida, Toshihiko, Williams, Keith, Kashiwagi, Keiko, Igarashi, Kazuei
• Format: Article
• Language: English
• Subjects: Aminoacid receptors (glycine, glutamate, gaba); aminoglycoside antibiotics; Aminoglycosides - pharmacology; Animals; Binding sites; Binding Sites - drug effects; Biochemistry; Biological and medical sciences; Biophysical Phenomena; Carbon Isotopes - metabolism; Cell receptors; Cell structures and functions; Central nervous system; Central neurotransmission. Neuromudulation. Pathways and receptors; Dose-Response Relationship, Drug; E coli; Electric Stimulation - methods; Escherichia coli; Excitatory Amino Acid Antagonists - pharmacology; Female; Fundamental and applied biological sciences. Psychology; ifenprodil; Membrane Potentials - drug effects; Membrane Potentials - physiology; Molecular and cellular biology; Neurology; Neurotransmitters; Oocytes - drug effects; Oocytes - metabolism; Oocytes - physiology; Patch-Clamp Techniques; Piperidines - pharmacology; Protein Conformation; Receptors, N-Methyl-D-Aspartate - chemistry; Receptors, N-Methyl-D-Aspartate - drug effects; Recombinant Proteins - metabolism; regulatory domain of N‐methyl‐d‐aspartate receptor; spermine; Spermine - pharmacology; Time Factors; Tritium - metabolism; Vertebrates: nervous system and sense organs; Xenopus laevis
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2008.05729.x

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.