title: An allosteric interaction between the NMDA receptor polyamine and ifenprodil sites in rat cultured cortical neurones.
abstract: 1. The atypical NR2B subunit-selective NMDA receptor antagonist ifenprodil was originally believed to act as a competitive antagonist at the polyamine binding site of the NMDA receptor. However, a number of studies have suggested that ifenprodil might bind to a distinct site. 2. Using whole-cell voltage clamp recordings, we have studied the interaction of spermine with both ifenprodil and the related NR2B selective antagonist Ro 8-4304 at the NMDA receptor in rat cultured cortical neurones in the presence of saturating concentrations of glycine. 3. Ifenprodil and Ro 8-4304 inhibited steady-state currents evoked by 100 microM NMDA in the absence of spermine with IC50 values of 0.3 and 0.6 microM, respectively. In the presence of 1 and 3 mM spermine, IC50 values for ifenprodil were 1.4 and 1.8 microM and for Ro 8-4304 they were 3. 0 and 7.5 microM, respectively. 4. In the presence of spermine, the on-time constant of receptor blockade by both antagonists was significantly slower than control and the off-time constant of recovery from receptor blockade following removal of Ro 8-4304 was significantly faster. 5. Fast application of spermine during an NMDA steady-state current in the continuous presence of a subsaturating concentration of either antagonist resulted in a biphasic increase in the current, consistent with a fast increase upon spermine binding and a slow increase resultant from dissociation of antagonist due to spermine binding-induced allosteric reduction in receptor antagonist affinity. In agreement with this, at higher, saturating concentrations of antagonist, the slow increase in current amplitude was markedly reduced or absent. 6. These observations are consistent with a non-competitive, allosteric interaction between spermine and the antagonists, such that spermine binding to the NMDA receptor results in a reduction in receptor affinity for the antagonists and vice versa. 7. The effects of Mg2+ on the NMDA-evoked currents and its interaction with ifenprodil were similar to those of spermine, supporting the suggestion that Mg2+ might be the physiological ligand acting at the spermine site mediating glycine-independent stimulation.
authors.affiliation: Pharma Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland.
authors.forename: J N
authors.initials: JN
authors.lastname: Kew
authors.forename: J A
authors.initials: JA
authors.lastname: Kemp
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D001665:Binding Sites; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology*; D004622:Embryo, Mammalian; D005071:Evoked Potentials / Q000187:drug effects; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000502:physiology*; D013096:Spermine / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C111258:4-(3-(4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl)-2-hydroxy-propoxy)-benzamide; D001549:Benzamides; D018691:Excitatory Amino Acid Antagonists; D010880:Piperidines; D011725:Pyridines; D016194:Receptors, N-Methyl-D-Aspartate; D013096:Spermine; D016202:N-Methylaspartate; D008274:Magnesium; C010739:ifenprodil