title: Low concentrations of neuroactive steroids alter kinetics of [3H]ifenprodil binding to the NMDA receptor in rat frontal cortex.
abstract: The modulatory effects of the two neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3alpha5betaS) on [3H]ifenprodil binding to the N-methyl-D-aspartate (NMDA) receptor in rat frontal cortex were studied. The binding for [3H]ifenprodil itself displayed monophasic kinetics in all experiments. None of the neurosteroids displaced the radioligand from its binding site on the NR2B subunit of the NMDA receptor. However, their continual presence at nanomolar concentrations had significant effects on ligand binding kinetics, interacting through distinct sites in saturation, competition and dissociation experiments. PS at 30 nM enhanced the specific binding to about 150% of that in its absence and enhanced the dissociation rate three-fold indicating a positive modulation of [3H]ifenprodil binding to the NMDA receptor. Furthermore, PS increased Bmax and decreased Kd suggesting that the neurosteroid exposes new [3H]ifenprodil binding sites with altered properties. In contrast, 3alpha5betaS (30 nM) decreased specific [3H]ifenprodil binding to approximately 40% of that determined for the radioligand alone. The presence of 3alpha5betaS at nanomolar concentrations induced biphasic curve fits in saturation, competition as well as dissociation experiments. In conclusion, the present study show that the allosteric modulators PS or 3alpha5betaS change [3H]ifenprodil binding kinetics in a way indicating conformational alteration of its binding site on the NR2B subunit.
authors.affiliation: Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, S-751 24 Uppsala, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Johansson
authors.forename: Per-Anders
authors.initials: PA
authors.lastname: Frändberg
authors.forename: Fred
authors.initials: F
authors.lastname: Nyberg
authors.forename: Pierre
authors.initials: P
authors.lastname: Le Grevès
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005625:Frontal Lobe / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010880:Piperidines / Q000378:metabolism*; D011280:Pregnanolone / Q000378:metabolism / Q000494:pharmacology*; D011284:Pregnenolone / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D014316:Tritium
chemical_list: C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; C018370:pregnenolone sulfate; D014316:Tritium; D011284:Pregnenolone; D011280:Pregnanolone; C010739:ifenprodil