Low concentrations of neuroactive steroids alter kinetics of [ 3 H]ifenprodil binding to the NMDA receptor in rat frontal cortex

The modulatory effects of the two neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3 α 5 β S) on [ 3 H]ifenprodil binding to the N ‐methyl‐ D ‐aspartate (NMDA) receptor in rat frontal cortex were studied. The binding for [ 3 H]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 n M enhanced the specific binding to about 150% of that in its absence and enhanced the dissociation rate three‐fold indicating a positive modulation of [ 3 H]ifenprodil binding to the NMDA receptor. Furthermore, PS increased B max and decreased K d suggesting that the neurosteroid exposes new [ 3 H]ifenprodil binding sites with altered properties. In contrast, 3 α 5 β S (30 n M ) decreased specific [ 3 H]ifenprodil binding to approximately 40% of that determined for the radioligand alone. The presence of 3 α 5 β S 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 3 α 5 β S change [ 3 H]ifenprodil binding kinetics in a way indicating conformational alteration of its binding site on the NR2B subunit.British Journal of Pharmacology (2005) 146 , 894–902. doi: 10.1038/sj.bjp.0706397