Development of a selective competitive receptor binding assay for the determination of the affinity to NR2B containing NMDA receptors.
A selective, rapid and efficient competitive binding assay for the determination of the affinity of compounds towards the ifenprodil binding site of NR2B subunit containing NMDA receptors has been developed. In the assay system, [(3)H]ifenprodil was used as radioligand and membrane homogenates from L(tk-) cells stably expressing recombinant human NR1a/NR2B receptors served as the receptor material. Sonication of the cells during work-up, performing all steps with 96-well multiplates and using a solid scintillator represent particular features of this assay. The binding kinetics was investigated by saturation and association/dissociation experiments. [(3)H]ifenprodil bound to a single, saturable site on human recombinant NR1a/NR2B receptors, resulting in a B(max)-value of 25.8 pmol/microg protein and K(d)-value of 7.6+/-2.3 nM (SEM). The dissociation rate constant (k(off)) was 0.03861 min(-1) and the association rate constant k(on) resulted in 0.00625 nM(-1)min(-1). The specificity of the assay was proved with cells not treated with dexamethasone, which has to be added to induce NMDA receptor synthesis of the cells. Additionally, the absence of alpha(1), sigma(1) and sigma(2) receptors was shown. The K(i)-values of the NR2B ligands ifenprodil and eliprodil determined with the new assay are in good accordance with reported data.
Schepmann
Dirk
D
Fachbereich Chemie und Pharamzie, Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Hittorfstrasse 58-62, D-48149 Münster, Germany.
Frehland
Bastian
B
Lehmkuhl
Kirstin
K
Tewes
Bastian
B
Wünsch
Bernhard
B
D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D010880:Piperidines / Q000378:metabolism*; D011869:Radioligand Assay; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; C010739:ifenprodil