N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

• Keywords
• Channelopathy, Endogenous neuroactive steroid, GRIN2 genes, Glutamate receptors, Rescue pharmacology, Surface expression,
• MeSH
• Electrophysiological Phenomena MeSH
• Humans MeSH
• Neurosteroids * MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Neurosteroids * MeSH
• NR2B NMDA receptor MeSH Browser
• Receptors, N-Methyl-D-Aspartate * MeSH

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5β-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5β-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5β-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5β-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

• Keywords
• glutamate, negative allosteric modulator, steroid, thigmotaxis, zebrafish,
• MeSH
• Zebrafish MeSH
• Esters MeSH
• gamma-Aminobutyric Acid MeSH
• Rats MeSH
• Glutamic Acid MeSH
• Pregnanolone * pharmacology   chemistry   MeSH
• Receptors, GABA-A MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Esters MeSH
• gamma-Aminobutyric Acid MeSH
• Glutamic Acid MeSH
• Pregnanolone * MeSH
• Receptors, GABA-A MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

BACKGROUND AND PURPOSE: Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG. EXPERIMENTAL APPROACH: We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation. KEY RESULTS: Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets. CONCLUSION AND IMPLICATIONS: We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.

• Keywords
• Munc13-1, glutamate, neurosteroid, phorbol ester, pregnanolone, presynaptic,
• MeSH
• HEK293 Cells MeSH
• Rats MeSH
• Glutamic Acid MeSH
• Humans MeSH
• Neurosteroids * MeSH
• Pregnanolone * pharmacology   MeSH
• Sulfates MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glutamic Acid MeSH
• Neurosteroids * MeSH
• Pregnanolone * MeSH
• Sulfates MeSH

NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

• Keywords
• NMDAR, carboxy-terminal domain, molecular dynamics simulation, neurosteroid, palmitoylation, single-channel recording,
• MeSH
• HEK293 Cells MeSH
• Hippocampus physiology   MeSH
• Rats MeSH
• Humans MeSH
• Lipoylation drug effects   physiology   MeSH
• Neuroprotection physiology   MeSH
• Rats, Wistar MeSH
• Pregnanes metabolism   pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 20-oxo-5beta-pregnan-3alpha-yl sulfate MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnanes MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

• Keywords
• glutamate receptors, neurosteroids, patch clamp, structure,
• MeSH
• Alanine genetics   MeSH
• Cell Membrane drug effects   MeSH
• Cholesterol metabolism   MeSH
• Electrophysiological Phenomena MeSH
• HEK293 Cells MeSH
• Protein Conformation MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Pregnenolone pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate drug effects   MeSH
• Molecular Dynamics Simulation MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• Cholesterol MeSH
• NMDA receptor A1 MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnenolone MeSH
• pregnenolone sulfate MeSH Browser
• Receptors, N-Methyl-D-Aspartate MeSH

UNLABELLED: 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 STATEMENT: Synaptic 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.

• Keywords
• NMDA receptor, memantine, neuroprotection, neurosteroid, pregnanolone sulfate, synaptic transmission,
• MeSH
• Excitatory Postsynaptic Potentials drug effects   MeSH
• HEK293 Cells MeSH
• Hippocampus metabolism   MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Synaptic Transmission drug effects   MeSH
• Neuroprotective Agents pharmacology   MeSH
• Motor Activity drug effects   MeSH
• Rats, Wistar MeSH
• Pregnanolone chemistry   pharmacology   MeSH
• Pregnanes chemistry   pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate antagonists & inhibitors   drug effects   genetics   MeSH
• Avoidance Learning drug effects   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Neuroprotective Agents MeSH
• NR2B NMDA receptor MeSH Browser
• Pregnanolone MeSH
• Pregnanes MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

• MeSH
• Amino Acid Motifs MeSH
• Humans MeSH
• Mutation * MeSH
• Pregnanolone * chemistry   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Vestibule, Labyrinth * chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Pregnanolone * MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

BACKGROUND AND PURPOSE: NMDA receptors are glutamatergic ionotropic receptors involved in excitatory neurotransmission, synaptic plasticity and excitotoxic cell death. Many allosteric modulators can influence the activity of these receptors positively or negatively, with behavioural consequences. 20-Oxo-5β-pregnan-3α-yl sulphate (pregnanolone sulphate; PA-6) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective. We tested the hypothesis that the interaction of PA-6 with the plasma membrane is critical for its inhibitory effect at NMDA receptors. EXPERIMENTAL APPROACH: Electrophysiological recordings and live microscopy were performed on heterologous HEK293 cells expressing GluN1/GluN2B receptors and cultured rat hippocampal neurons. KEY RESULTS: Our experiments showed that the kinetics of the steroid inhibition were slow and not typical of drug-receptor interaction in an aqueous solution. In addition, the recovery from steroid inhibition was accelerated by β- and γ-cyclodextrin. Values of IC(50) assessed for novel synthetic C3 analogues of PA-6 differed by more than 30-fold and were positively correlated with the lipophilicity of the PA-6 analogues. Finally, the onset of inhibition induced by C3 analogues of PA-6 ranged from use-dependent to use-independent. The onset and offset of cell staining by fluorescent analogues of PA-6 were slower than those of steroid-induced inhibition of current responses mediated by NMDA receptors. CONCLUSION AND IMPLICATIONS: We conclude that steroid accumulation in the plasma membrane is the route by which it accesses a binding site on the NMDA receptor. Thus, our results provide a possible structural framework for pharmacologically targeting the transmembrane domains of the receptor.

• MeSH
• Action Potentials drug effects   MeSH
• Excitatory Amino Acid Antagonists chemistry   pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Microscopy, Fluorescence MeSH
• HEK293 Cells MeSH
• Rats MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Neurons drug effects   metabolism   MeSH
• Neuroprotective Agents chemistry   pharmacology   MeSH
• Neurotransmitter Agents chemistry   pharmacology   MeSH
• Pregnanes chemistry   pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate antagonists & inhibitors   genetics   MeSH
• Transfection MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 20-oxo-5beta-pregnan-3alpha-yl sulfate MeSH Browser
• Excitatory Amino Acid Antagonists MeSH
• Neuroprotective Agents MeSH
• Neurotransmitter Agents MeSH
• Pregnanes MeSH
• Receptors, N-Methyl-D-Aspartate MeSH