Characterisation of N‐Methyl‐D‐Aspartate Receptor‐Specific [3H]Ifenprodil Binding to Recombinant Human NR1a/NR2B Receptors Compared with Native Receptors in Rodent Brain Membranes

We have performed [ 3 H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [ 3 H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B max values of 1.83 and 2.45 pmol/mg of protein, respectively, and K D values of 33.5 and 24.8 nM, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R * ,R * )-4-hydroxy-α-(4-hydroxyphenyl)-β-methyl-4-phenyl-1-piperidineethanol [(±)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3,4-dihydro-2H-1-benzopyran-4,7-diol [(±)-CP-283,097], and (R * ,S * )-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol [(±)-Ro 25-6981] was very similar for inhibition of [3H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [ 3 H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [ 3 H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [ 3 H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (±)-CP-101,606, (±)-CP-283,097, and (±)-Ro 25-6981] given systemically.