Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N-methyl-D-aspartate receptor.
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 K(d) values for binding of [(14)C]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 microM, respectively. [(3)H]Ifenprodil bound to NR1-R (K(d), 0.18 microM) and NR2B-R (K(d), 0.21 microM), but not to NR2A-R at the concentrations tested (0.1-0.8 microM). These K(d) values were confirmed by circular dichroism measurements. The K(d) 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.
Han
Xia
X
Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Tomitori
Hideyuki
H
Mizuno
Satomi
S
Higashi
Kyohei
K
Füll
Christine
C
Fukiwake
Tomohide
T
Terui
Yusuke
Y
Leewanich
Pathama
P
Nishimura
Kazuhiro
K
Toida
Toshihiko
T
Williams
Keith
K
Kashiwagi
Keiko
K
Igarashi
Kazuei
K
D000617:Aminoglycosides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D055592:Biophysical Phenomena; D002247:Carbon Isotopes / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology*; D011487:Protein Conformation; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000187:drug effects*; D011994:Recombinant Proteins / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology*; D013997:Time Factors; D014316:Tritium / Q000378:metabolism; D014982:Xenopus laevis
D000617:Aminoglycosides; D002247:Carbon Isotopes; D018691:Excitatory Amino Acid Antagonists; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D014316:Tritium; D013096:Spermine; C010739:ifenprodil