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

Abstract

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-5β-pregnan-3α-yl sulfate (3α5βS) action at NMDA receptors. 3α5βS 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 3α5βS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3α5βS 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 3α5βS at NMDA receptors. In accordance with the model, 3α5βS 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 3α5βS 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. Copyright © 2005 Society for Neuroscience.