Conformational basis of subtype-specific allosteric control of NMDA receptor gating.

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that are integral to synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties, which support their individual physiological roles in the nervous system. To understand the conformational basis of this diversity, we assessed the conformation of the common GluN1 subunit in receptors with different GluN2 subunits using single-molecule fluorescence resonance energy transfer (smFRET). We established smFRET sensors in the ligand binding domain and modulatory amino-terminal domain to study an apo-like state and partially liganded activation intermediates, which have been elusive to structural analysis. Our results demonstrate a strong, subtype-specific influence of apo and glutamate-bound GluN2 subunits on GluN1 rearrangements, suggesting a conformational basis for the highly divergent levels of receptor activity, desensitization and agonist potency. Chimeric analysis reveals structural determinants that contribute to the subtype differences. Our study provides a framework for understanding GluN2-dependent functional properties and could open new avenues for subtype-specific modulation.

Effects of Pregnanolone Glutamate and Its Metabolites on GABAA and NMDA Receptors and Zebrafish Behavior.

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-5ß-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-5ß-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5ß-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5ß-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.

Pregnane-based steroids are novel positive NMDA receptor modulators that may compensate for the effect of loss-of-function disease-associated GRIN mutations.

BACKGROUND AND PURPOSE: N-methyl-D-aspartate receptors (NMDARs) play a critical role in synaptic plasticity, and mutations in human genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. Compounds with a positive allosteric effect are thought to compensate for reduced receptor function. EXPERIMENTAL APPROACH: We have used whole-cell patch-clamp electrophysiology on recombinant rat NMDARs and human variants found in individuals with neuropsychiatric disorders, in combination with in silico modelling, to explore the site of action of novel epipregnanolone-based NMDAR modulators. KEY RESULTS: Analysis of the action of 4-(20-oxo-5ß-pregnan-3ß-yl) butanoic acid (EPA-But) at the NMDAR indicates that the effect of this steroid with a "bent" structure is different from that of cholesterol and oxysterols and shares a disuse-dependent mechanism of NMDAR potentiation with the "planar" steroid 20-oxo-pregn-5-en-3ß-yl sulfate (PE-S). The potentiating effects of EPA-But and PE-S are additive. Alanine scan mutagenesis identified residues that reduce the potentiating effect of EPA-But. No correlation was found between the effects of EPA-But and PE-S at mutated receptors that were less sensitive to either steroid. The relative degree of potentiation induced by the two steroids also differed in human NMDARs carrying rare variants of hGluN1 or hGluN2B subunits found in individuals with neuropsychiatric disorders, including intellectual disability, epilepsy, developmental delay, and autism spectrum disorder. CONCLUSION AND IMPLICATIONS: Our results show novel sites of action for pregnanolones at the NMDAR and provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with glutamatergic system hypofunction.

Pitfalls of NMDA Receptor Modulation by Neuroactive Steroids. The Effect of Positive and Negative Modulation of NMDA Receptors in an Animal Model of Schizophrenia.

Evidence from clinical and preclinical studies implicates dysfunction of N-methyl-D-aspartate receptors (NMDARs) in schizophrenia progression and symptoms. We investigated the antipsychotic effect of two neuroactive steroids in an animal model of schizophrenia induced by systemic application of MK-801. The neuroactive steroids differ in their mechanism of action at NMDARs. MS-249 is positive, while PA-Glu is a negative allosteric NMDAR modulator. We hypothesized that the positive NMDA receptor modulator would attenuate deficits caused by MK-801 co-application more effectively than PA-Glu. The rats were tested in a battery of tests assessing spontaneous locomotion, anxiety and cognition. Contrary to our expectations, PA-Glu exhibited a superior antipsychotic effect to MS-249. The performance of MS-249-treated rats in cognitive tests differed depending on the level of stress the rats were exposed to during test sessions. In particular, with the increasing severity of stress exposure, the performance of animals worsened. Our results demonstrate that enhancement of NMDAR function may result in unspecific behavioral responses. Positive NMDAR modulation can influence other neurobiological processes besides memory formation, such as anxiety and response to stress.

Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation.

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.

Palmitoylation Controls NMDA Receptor Function and Steroid Sensitivity.

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-5ß-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5ß-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.

Site of Action of Brain Neurosteroid Pregnenolone Sulfate at the N-Methyl-D-Aspartate Receptor.

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.

Conformational pathway provides unique sensitivity to a synaptic mGluR.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.

Genetically Targeted Optical Control of an Endogenous G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in biology. However, our understanding of their function in complex living systems is limited because we lack tools that can target individual receptors with sufficient precision. State-of-the-art approaches, including DREADDs, optoXRs, and PORTL gated-receptors, control GPCR signaling with molecular, cell type, and temporal specificity. Nonetheless, these tools are based on engineered non-native proteins that may (i) express at nonphysiological levels, (ii) localize and turnover incorrectly, and/or (iii) fail to interact with endogenous partners. Alternatively, membrane-anchored ligands (t-toxins, DARTs) target endogenous receptors with molecular and cell type specificity but cannot be turned on and off. In this study, we used a combination of chemistry, biology, and light to control endogenous metabotropic glutamate receptor 2 (mGluR2), a Family C GPCR, in primary cortical neurons. mGluR2 was rapidly, reversibly, and selectively activated with photoswitchable glutamate tethered to a genetically targeted-plasma membrane anchor (membrane anchored Photoswitchable Orthogonal Remotely Tethered Ligand; maPORTL). Photoactivation was tuned by adjusting the length of the PORTL as well as the expression level and geometry of the membrane anchor. Our findings provide a template for controlling endogenous GPCRs with cell type specificity and high spatiotemporal precision.

Strong Inhibitory Effect, Low Cytotoxicity and High Plasma Stability of Steroidal Inhibitors of N-Methyl-D-Aspartate Receptors With C-3 Amide Structural Motif.

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 µM) than PAG (IC50 = 51.7 µM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 µM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 µM and 276.4 ± 178.7 µM, respectively). In addition, compound 6 (10 µM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

Positive Modulators of the N-Methyl-d-aspartate Receptor: Structure-Activity Relationship Study of Steroidal 3-Hemiesters.

Here, we report the synthesis of pregn-5-ene and androst-5-ene dicarboxylic acid esters and explore the structure-activity relationship (SAR) for their modulation of N-methyl-d-aspartate receptors (NMDARs). All compounds were positive modulators of recombinant GluN1/GluN2B receptors (EC50 varying from 1.8 to 151.4 µM and Emax varying from 48% to 452%). Moreover, 10 compounds were found to be more potent GluN1/GluN2B receptor modulators than endogenous pregnenolone sulfate (EC50 = 21.7 µM). The SAR study revealed a relationship between the length of the residues at carbon C-3 of the steroid molecule and the positive modulatory effect at GluN1/GluN2B receptors for various D-ring modifications. A selected compound, 20-oxo-pregnenolone hemiadipate, potentiated native NMDARs to a similar extent as GluN1/GluN2A-D receptors and inhibited AMPARs and GABAAR responses. These results provide a unique opportunity for the development of new steroid based drugs with potential use in the treatment of neuropsychiatric disorders involving hypofunction of NMDARs.

Surface Expression, Function, and Pharmacology of Disease-Associated Mutations in the Membrane Domain of the Human GluN2B Subunit.

N-methyl-D-aspartate receptors (NMDARs), glutamate-gated ion channels, mediate signaling at the majority of excitatory synapses in the nervous system. Recent sequencing data for neurological and psychiatric patients have indicated numerous mutations in genes encoding for NMDAR subunits. Here, we present surface expression, functional, and pharmacological analysis of 11 de novo missense mutations of the human hGluN2B subunit (P553L; V558I; W607C; N615I; V618G; S628F; E657G; G820E; G820A; M824R; L825V) located in the pre-M1, M1, M2, M3, and M4 membrane regions. These variants were identified in patients with intellectual disability, developmental delay, epileptic symptomatology, and autism spectrum disorder. Immunofluorescence microscopy indicated that the ratio of surface-to-total NMDAR expression was reduced for hGluN1/hGluN2B(S628F) receptors and increased for for hGluN1/hGluN2B(G820E) receptors. Electrophysiological recordings revealed that agonist potency was altered in hGluN1/hGluN2B(W607C; N615I; and E657G) receptors and desensitization was increased in hGluN1/hGluN2B(V558I) receptors. The probability of channel opening of hGluN1/hGluN2B (V558I; W607C; V618G; and L825V) receptors was diminished ~10-fold when compared to non-mutated receptors. Finally, the sensitivity of mutant receptors to positive allosteric modulators of the steroid origin showed that glutamate responses induced in hGluN1/hGluN2B(V558I; W607C; V618G; and G820A) receptors were potentiated by 59-96% and 406-685% when recorded in the presence of 20-oxo-pregn-5-en-3ß-yl sulfate (PE-S) and androst-5-en-3ß-yl hemisuccinate (AND-hSuc), respectively. Surprisingly hGluN1/hGluN2B(L825V) receptors were strongly potentiated, by 197 and 1647%, respectively, by PE-S and AND-hSuc. Synaptic-like responses induced by brief glutamate application were also potentiated and the deactivation decelerated. Further, we have used homology modeling based on the available crystal structures of GluN1/GluN2B NMDA receptor followed by molecular dynamics simulations to try to relate the functional consequences of mutations to structural changes. Overall, these data suggest that de novo missense mutations of the hGluN2B subunit located in membrane domains lead to multiple defects that manifest by the NMDAR loss of function that can be rectified by steroids. Our results provide an opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with hypofunction of the glutamatergic system.

Total Synthesis of ent-Pregnanolone Sulfate and Its Biological Investigation at the NMDA Receptor.

A unique asymmetric total synthesis of the unnatural enantiomer of pregnanolone, as well as a study of its biological activity at the NMDA receptor, is reported. The asymmetry is introduced by a highly atom-economic organocatalytic Robinson annulation. A new method for the construction of the cyclopentane D-ring consisting of CuI-catalyzed conjugate addition and oxygenation followed by thermal cyclization employing the persistent radical effect was developed. ent-Pregnanolone sulfate is surprisingly only 2.6-fold less active than the natural neurosteroid.

Physicochemical and biological properties of novel amide-based steroidal inhibitors of NMDA receptors.

Herein, we report a new class of amide-based inhibitors (1-4) of N-methyl-d-aspartate receptors (NMDARs) that were prepared as analogues of pregnanolone sulfate (PAS) and pregnanolone glutamate (PAG) - the steroidal neuroprotective NMDAR inhibitors. A series of experiments were conducted to evaluate their physicochemical and biological properties: (i) the inhibitory effect of compounds 3 and 4 on NMDARs was significantly improved (IC50=1.0 and 1.4µM, respectively) as compared with endogenous inhibitor - pregnanolone sulfate (IC50=24.6µM) and pregnanolone glutamate (IC50=51.7µM); (ii) physicochemical properties (logP and logD) were calculated; (iii) Caco-2 assay revealed that the permeability properties of compounds 2 and 4 are comparable with pregnanolone glutamate; (iv) compounds 1-4 have minimal or no adverse hepatic effect; (v) compounds 1-4 cross blood-brain-barrier.

Neurosteroid-like Inhibitors of N-Methyl-d-aspartate Receptor: Substituted 2-Sulfates and 2-Hemisuccinates of Perhydrophenanthrene.

N-Methyl-d-aspartate receptors (NMDARs) display a critical role in various diseases of the central nervous system. The activity of NMDARs can be modulated by neurosteroids. Herein, we report a structure-activity relationship study for perhydrophenanthrene analogues possessing a framework that mimics the steroidal ring system. This study comprises the design, synthesis, and assessment of the biological activity of a library of perhydrophenanthrene 2-sulfates and 2-hemisuccinates (1-10). Their ability to modulate NMDAR-induced currents was tested on recombinant GluN1/GluN2B receptors. Our results demonstrate that such structural optimization leads to compounds that are inhibitors of NMDARs. Notably, compound 9 (IC50 = 15.6 µM) was assessed as a more potent inhibitor of NMDAR-induced currents than the known endogenous neurosteroid, pregnanolone sulfate (IC50 = 24.6 µM).

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives.

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.

A New Class of Potent N-Methyl-D-Aspartate Receptor Inhibitors: Sulfated Neuroactive Steroids with Lipophilic D-Ring Modifications.

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play a crucial role in excitatory synaptic transmission. However, the overactivation of NMDARs can lead to excitotoxic cell damage/death, and as such, they play a role in numerous neuropathological conditions. The activity of NMDARs is known to be influenced by a wide variety of allosteric modulators, including neurosteroids, which in turn makes them promising therapeutic targets. In this study, we describe a new class of neurosteroid analogues which possess structural modifications in the steroid D-ring region. These analogues were tested on recombinant GluN1/GluN2B receptors to evaluate the structure-activity relationship. Our results demonstrate that there is a strong correlation between this new structural feature and the in vitro activity, as all tested compounds were evaluated as more potent inhibitors of NMDA-induced currents (IC50 values varying from 90 nM to 5.4 µM) than the known endogeneous neurosteroid-pregnanolone sulfate (IC50 = 24.6 µM).

Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule.

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.

Cholesterol modulates open probability and desensitization of NMDA receptors.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-ß-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-ß-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.

Neuroactive steroids with perfluorobenzoyl group.

During an initial study in searching for the alternative derivatives suitable for photolabeling of neuroactive steroids, perfluorobenzoates and perfluorobenzamides in position 17 of 5ß-androstan-3α-ol were synthesized from the corresponding 17-hydroxy and 17-amino derivatives. After transformation into glutamates or sulfates, 17α-epimers had comparable inhibitory activity at NMDA receptors to the natural neurosteroid (20-oxo-5ß-pregnan-3ß-yl sulfate), however, were more potent (2- to 36-fold) than their 17ß-substituted analogs. In one case, fluorine in position 4' of perfluorobenzoate group was substituted with azide and activity of the final glutamate was retained comparing with the corresponding perfluorobenzoate. The series was expanded with perfluorobenzoyl derivatives of pregnanolone: Perfluorobenzamide of glutamate and perfluorobenzoate of 11α-hydroxy pregnanolone were prepared and tested. From nine tested compounds, four of them exhibit very good inhibition activity and can serve as promising leads for photolabeling experiments.