20-Oxo-5β-pregnan-3α-yl sulfate is a use-dependent NMDA receptor inhibitor

NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5 beta-pregnan-3 alpha-yl sulfate (3 alpha 5 beta S) action at NMDA receptors. 3 alpha 5 beta S was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3 alpha 5 beta S-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3 alpha 5 beta S in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3 alpha 5 beta S at NMDA receptors. In accordance with the model, 3 alpha 5 beta S was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3 alpha 5 beta S is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.