Molecular mechanisms for nanomolar concentrations of neurosteroids at NR1/NR2B receptors

Neurosteroids are endogenous steroids acting in the central nervous system. They participate in synaptic plasticity, memory and learning, Alzheimer's disease, and certain drug reward. Some mechanisms behind these effects are thought to be nongenomic, e.g., they modulate the function of the N-methyl-d-aspartate (NMDA) receptor complex. In this study, we used a Chinese hamster ovary cell line stably transfected with NMDA receptor constituents NR1/NR2B, to investigate the effects of nanomolar concentrations of the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate (3alpha5betaS) on binding of the radioligand [(3)H]ifenprodil. Neither of the steroids displaced [(3)H]ifenprodil, but both induced a shift in its fit from one to two binding sites. The effects of the neurosteroids were also measured as changes in intracellular calcium ([Ca(2+)](i)) after glutamate stimulation. Although the steroids did not alter the response to glutamate, they influenced the extent of ifenprodil blockade of the receptor: PS increased and 3alpha5betaS decreased this effect. The coincubation of several NMDA receptor ligands in the assay indicated that PS and 3alpha5betaS act via different binding sites from those for glutamate, glycine, and dithiothreitol. Combining the two steroids revealed that they do not share a common binding site. In conclusion, these results substantiate previous evidence of the allosteric modulatory effect induced by PS and 3alpha5betaS on NMDA receptors at nanomolar concentrations. The neurosteroid-mediated modulation of the receptor is also reflected in an altered glutamate stimulated [Ca(2+)](i), in response to ifenprodil.