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 [H-3]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, [H-3]lfenprodil 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-alpha-(4-hydroxyphenyl)-beta -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*)-alpha-(4-hydroxyphenyl)-beta -methyl-4-(phenylmethyl)-1-piperidinepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [H-3]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [H-3]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 [H-3]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors, The NMDA receptor-specific [H-3]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.