An allosteric interaction between the NMDA receptor polyamine and ifenprodil sites in rat cultured cortical neurones

Authors


Corresponding author J. A. Kemp: Pharma Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd, Building 69/412, CH-4070 Basel, Switzerland. Email: john.kemp@roche.com

Abstract

  • 1The 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.
  • 2Using 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.
  • 3Ifenprodil and Ro 8-4304 inhibited steady-state currents evoked by 100 μm NMDA in the absence of spermine with IC50 values of 0.3 and 0.6 μm, respectively. In the presence of 1 and 3 mm spermine, IC50 values for ifenprodil were 1.4 and 1.8 μm and for Ro 8-4304 they were 3.0 and 7.5 μm, respectively.
  • 4In 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.
  • 5Fast 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.
  • 6These 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.
  • 7The 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.

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