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

We have performed [ 3 H]ifenprodil bindingexperiments under NMDA receptor‐specific assay conditions to provide the firstdetailed characterisation of the pharmacology of the ifenprodil site on NMDANR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stablyexpressed in L(tk‐) cells, in comparison with rat cortex/hippocampusmembranes. [ 3 H]Ifenprodil bound to a single, saturable site on bothhuman 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 n M ,respectively. The affinity of various ifenprodil site ligands—eliprodil,( R * , R * )‐4‐hydroxy‐α‐(4‐hydroxyphenyl)‐β‐methyl‐4‐pehnyl‐1‐piperidineethanol[(±)‐CP‐101,606], cis ‐3‐[4‐(4‐fluorophenyl)‐4‐hydroxy‐1‐piperidinyl]‐3,4‐dihydro‐2 H ‐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[ 3 H]ifenprodil binding to recombinant human NR1a/NR2B and nativerat receptors, whereas allosteric inhibition of [ 3 H]ifenprodilbinding by polyamine site ligands (spermine, spermidine, and arcaine) showedapproximately twofold lower affinity for recombinant receptors compared withnative receptors. Glutamate site ligands were less effective at modulating[ 3 H]ifenprodil binding to recombinant NR1a/NR2B receptors comparedwith native rat receptors. The NMDA receptor‐specific[ 3 H]ifenprodil binding conditions described were also applied to exvivo experiments to determine the receptor occupancy of ifenprodil siteligands [ifenprodil, (±)‐CP‐101,606, (±)‐CP‐283,097, and(±)‐Ro 25‐6981] given systemically.