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Pregnanolone sulfate promotes desensitization of activated NMDA receptors.

ABSTRACT: Neurosteroids are potent neuromodulators which act in part by binding to and modifying the activity of neurotransmitter-gated channels. Pregnanolone sulfate (PAS) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective in vivo. To delineate the mechanism of NMDA receptor inhibition by pregnanolone sulfate we used kinetic analyses of equilibrium single-channel currents recorded from individual GluN1/GluN2A receptors. Results show that PAS (0.1 mM) reduces single-channel open probability by 50% solely by increasing approximately 5-fold the mean time spent by receptors in closed conformations. From these data we derive a kinetic scheme that summarizes the effects of PAS on single channel kinetics, accounts for the PAS effects on macroscopic responses and leads us to propose that PAS inhibits NMDA receptor activity by shifting active receptors into desensitized conformations. These findings highlight the neurosteroid inhibitory site on NMDA receptors as a valuable therapeutic target against excitotoxic pathologies including acute and chronic neurodegeneration.

SUBMITTER: Kussius CL

PROVIDER: S-EPMC3150747 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Pregnanolone sulfate promotes desensitization of activated NMDA receptors.

Kussius Cassandra L CL Kaur Navjot N Popescu Gabriela K GK

The Journal of neuroscience : the official journal of the Society for Neuroscience 20090501 21

Neurosteroids are potent neuromodulators which act in part by binding to and modifying the activity of neurotransmitter-gated channels. Pregnanolone sulfate (PAS) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective in vivo. To delineate the mechanism of NMDA receptor inhibition by pregnanolone sulfate we used kinetic analyses of equilibrium single-channel currents recorded from individual GluN1/GluN2A receptors. Results show that PAS (0.1 mM) reduces single-channel ...[more]

PMID: 19474309

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Project description:Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential NMDAR inhibition depending on subcellular location has not been investigated systematically. We integrated recordings from heterologously expressed single NMDAR subtypes, kinetic modeling, and recordings of synaptically evoked NMDAR responses in acute brain slices to investigate mechanisms by which channel blockers may distinguish NMDAR subpopulations. We found that memantine and ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca2+-dependent desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells and of native synaptic NMDARs in cortical pyramidal neurons from mice of either sex increased in conditions that enhanced intracellular Ca2+ accumulation. Therefore, differential inhibition by memantine and ketamine based on NMDAR location is likely to result from location dependence of the intensity and duration of NMDAR activation. Modulation of Ca2+-dependent NMDAR desensitization is an unexplored mechanism of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather than specific receptor types, may be a viable strategy for future drug development.SIGNIFICANCE STATEMENT Memantine and ketamine are NMDA receptor (NMDAR) channel-blocking drugs with divergent clinical effects. Understanding mechanistically their differential actions may advance our understanding of nervous system disorders and suggest strategies for the design of more effective drugs. Here, we show that memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered after accumulation of intracellular Ca2+, a novel inhibitory mechanism. These properties may contribute to inhibition of distinct NMDAR subpopulations by memantine and ketamine and help to explain their differential clinical effects. Our results suggest stabilization of Ca2+-dependent desensitized states as a new strategy for pharmaceutical neuroprotection.

Project description:Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action.Significance statementSynaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

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Pregnanolone sulfate promotes desensitization of activated NMDA receptors.

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Pregnanolone sulfate promotes desensitization of activated NMDA receptors.

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