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

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Abstract
We have performed [3H]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. [3H]Ifenprodil bound to a single, saturable site on bothhuman recombinant NR1a/NR2B receptors and native rat receptors withBmax values of 1.83 and 2.45 pmol/mg of protein,respectively, and KD 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‐pehnyl‐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 nativerat receptors, whereas allosteric inhibition of [3H]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[3H]ifenprodil binding to recombinant NR1a/NR2B receptors comparedwith native rat receptors. The NMDA receptor‐specific[3H]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.

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