Intrasubunit and Intersubunit Steroid Binding Sites Independently and Additively Mediate α1ß2γ2L GABAA Receptor Potentiation by the Endogenous Neurosteroid Allopregnanolone.

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABAA receptor. The sites are located in the membrane-spanning domains of the receptor at the ß-α subunit interface (site I) and within the α (site II) and ß subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1ß2γ2L GABAA receptor by the endogenous neurosteroid allopregnanolone (3α5αP). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3α5αP, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3α5αP act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ-aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site.

Analysis of Modulation of the ρ1 GABAA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5ß-reduced steroids, sulfated or carboxylated steroids, and ß-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5ßP), 3α5ßP sulfate, and ß-estradiol. The site for 3α5ßP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor.

Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABAA receptors (GABAAR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABAAR-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11ß-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAAR PAM, to characterize steroid-binding sites in the Torpedo α2ßγδ nAChR in its native membrane environment. We found that F4N3Bzoxy-AP (IC50 = 31 µm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [3H]tenocyclidine to nAChRs in the desensitized state. At 300 µm, neither steroid inhibited binding of [3H]tetracaine, a closed-state selective channel blocker, or of [3H]acetylcholine. Photolabeling identified three distinct [3H]F4N3Bzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of ßVal-261 and δVal-269 (position M2-13'); 2) at the interface between the αM1 and αM4 helices, identified by photolabeling in αM1 (αCys-222/αLeu-223); and 3) at the lipid-protein interface involving γTrp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and αM1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroid-binding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists.

3ß-Methyl-Neurosteroid Analogs Are Preferential Positive Allosteric Modulators and Direct Activators of Extrasynaptic δ-Subunit γ-Aminobutyric Acid Type A Receptors in the Hippocampus Dentate Gyrus Subfield.

Neurosteroids are powerful modulators of γ-aminobutyric acid (GABA)-A receptors. Ganaxolone (3α-hydroxy-3ß-methyl-5α-pregnan-20-one, GX) and synthetic analogs of the neurosteroid allopregnanolone (AP) are designed to treat epilepsy and related conditions. However, their precise mechanism of action in native neurons remains unclear. Here, we sought to determine the mode of action of GX and its analogs at GABA-A receptors in native hippocampal neurons by analyzing extrasynaptic receptor-mediated tonic currents and synaptic receptor-mediated phasic currents. Concentration-response profiles of GX were determined in two cell types: δ-containing dentate gyrus granule cells (DGGCs) and γ2-containing CA1 pyramidal cells (CA1PCs). GX produced significantly greater potentiation of the GABA-A receptor-activated chloride currents in DGGCs (500%) than CA1PCs (200%). In the absence of GABA, GX evoked 2-fold greater inward currents in DGGCs than CA1PCs, which were 2-fold greater than AP within DGGCs. In hippocampus slices, GX potentiated and directly activated tonic currents in DGGCs. These responses were significantly diminished in DGGCs from δ-subunit knockout (δKO) mice, confirming GX's selectivity for δGABA-A receptors. Like AP, GX potentiation of tonic currents was prevented by protein kinase C inhibition. Furthermore, GX's protection against hippocampus-kindled seizures was significantly diminished in δKO mice. GX analogs exhibited greater potency and efficacy than GX on δGABA-A receptor-mediated tonic inhibition. In summary, these results provide strong evidence that GX and its analogs are preferential allosteric modulators and direct activators of extrasynaptic δGABA-A receptors regulating network inhibition and seizures in the dentate gyrus. Therefore, these findings provide a mechanistic rationale for the clinical use of synthetic neurosteroids in epilepsy and seizure disorders.

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.

Antispasmodic, bronchodilator, vasorelaxant and cardiosuppressant effects of Buxus papillosa.

BACKGROUND: The present research was carried out to investigate pharmacological properties of Buxus papillosa C.K. Schneid. (Buxaceae). METHODS: Buxus papillosa extracts of leaves (BpL), stem (BpS), roots (BpR) and BpL fractions: hexane (BpL-H), aqueous (BpL-A) also plant constituent, cyclomicrobuxine effect were studied in jejunum, atria, aorta and tracheal preparations from rabbit and guine-peg. RESULTS: Ca++ antagonistic effect of BpS, BpR, BpL-H, BpL-A and cyclomicrobuxine were conclusively suggested, when spontaneous contractions of rabbit jejunal preparation was relaxed along with subsequent relaxation of potassium chloride (80 mM) induced contractions. Ca++ antagonistic effect was further confirmed, when a prominent right shift like that of verapamil was observed in Ca++ concentration-response curves, drawn in a tissue pretreated with BpL (0.3-1.0 mg/mL). In rabbit tracheal tissues BpL, BpS, BpR, BpL-H and BpL-A produced a prominent relaxation in contractions induced by potassium chloride (80 mM) and carbachol (1 µm). When tested in rabbit aortic rings, BpL, BpS, BpR, BpL-H and BpL-A showed concentration-dependent (0.1-3.0 mg/mL) vasorelaxant effect against phenylephrine (1 µM) and high K+-induced contractions. In isolated guinea-pig right atria, BpL, BpS, BpR, BpL-H and BpL-A suppressed atrial force of spontaneous contractions, with BpL-A being most potent. CONCLUSIONS: Our results reveal that Buxus papillosa possesses gut, airways and cardiovascular inhibitory actions.

Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study.

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study.

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr-/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator.

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 µM), SGE-872 is selective for tonic, extrasynaptic α4ß3δ-containing GABAA receptors over typical synaptic α1ß2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.

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.

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ß3 subunit of the GABA(A) receptor.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5ß)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-ß3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-ß3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 µM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-ß3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the ß3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3ß,5ß)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-ß3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ß3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of ß-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-ß-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z = 1287.77), a ß-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and ß-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.

Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.

Inhibitory neurotransmission mediated by GABA(A) receptors can be modulated by the endogenous neurosteroids, allopregnanolone and tetrahydro-deoxycorticosterone. Neurosteroids are synthesized de novo in the brain during stress, pregnancyand after ethanol consumption, and disrupted steroid regulation of GABAergic transmission is strongly implicated in several debilitating conditions such as panic disorder, major depression, schizophrenia, alcohol dependence and catamenial epilepsy. Determining how neurosteroids interact with the GABA(A) receptor is a prerequisite for understanding their physiological and pathophysiological roles in the brain. Here we identify two discrete binding sites in the receptor's transmembrane domains that mediate the potentiating and direct activation effects of neurosteroids. They potentiate GABA responses from a cavity formed by the alpha-subunit transmembrane domains, whereas direct receptor activation is initiated by interfacial residues between alpha and beta subunits and is enhanced by steroid binding to the potentiation site. Thus, significant receptor activation by neurosteroids relies on occupancy of both the activation and potentiation sites. These sites are highly conserved throughout the GABA(A )receptor family, and their identification provides a unique opportunity for the development of new therapeutic, neurosteroid-based ligands and transgenic disease models of neurosteroid dysfunction.

Neurosteroid Modulation of GABAA Receptor Function by Independent Action at Multiple Specific Binding Sites.

Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epiallopregnanolone (3ß-OH) are both prevalent in the mammalian brain and produce opposite effects on GABAA receptor function, acting as positive and negative allosteric modulators, respectively. This Perspective provides a model to explain the actions of 3α-OH and 3ß-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABAA receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.

Microglial Cell Morphology and Phagocytic Activity Are Critically Regulated by the Neurosteroid Allopregnanolone: A Possible Role in Neuroprotection.

Microglial cells are key players in neural pathogenesis and microglial function regulation appears to be pivotal in controlling neuroinflammatory/neurological diseases. Here, we investigated the effects and mechanism of action of neurosteroid allopregnanolone (ALLO) on murine microglial BV-2 cells and primary microglia in order to determine ALLO-induced immunomodulatory potential and to provide new insights for the development of both natural and safe neuroprotective strategies targeting microglia. Indeed, ALLO-treatment is increasingly suggested as beneficial in various models of neurological disorders but the underlying mechanisms have not been elucidated. Therefore, the microglial cells were cultured with various serum concentrations to mimic the blood-brain-barrier rupture and to induce their activation. Proliferation, viability, RT-qPCR, phagocytosis, and morphology analyzes, as well as migration with time-lapse imaging and quantitative morphodynamic methods, were combined to investigate ALLO actions on microglia. BV-2 cells express subunits of GABA-A receptor that mediates ALLO activity. ALLO (10µM) induced microglial cell process extension and decreased migratory capacity. Interestingly, ALLO modulated the phagocytic activity of BV-2 cells and primary microglia. Our results, which show a direct effect of ALLO on microglial morphology and phagocytic function, suggest that the natural neurosteroid-based approach may contribute to developing effective strategies against neurological disorders that are evoked by microglia-related abnormalities.

Allosteric modulation of the NMDA receptor by neurosteroids in rat brain and the impact of long term morphine administration.

This study examined the allosteric modulation of the NMDA receptor by nanomolar concentrations of neurosteroids in rats treated long term with morphine. The neurosteroids dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PS) and pregnanolone sulfate (3α5ßS) are important mediators in the central nervous system. They induce rapid responses by non-classical steroidal mechanisms, e.g. via interaction with the N-methyl-D-aspartate (NMDA) receptor, and are known to modify the binding of ifenprodil to the NMDA receptor subunit NR2B. The NMDA receptor is involved in several processes, including memory, learning, synaptic plasticity and neuronal development. Morphine, a µ-opioid receptor agonist, has an important role in the clinical treatment of pain. The main drawback of morphine treatment is the associated development of dependence and tolerance. The mechanisms behind these phenomena are still to be elucidated, but several reports suggest the involvement of the NMDA receptor. The results of the present study indicate that the allosteric modulation induced by the neurosteroids DHEAS, PS and 3α5ßS was similar in all tested brain regions. This suggests that the NR2B receptor subunit behaves independently of its site of expression. Moreover, the NR2B subunit was up-regulated in the frontal cortex but not in the hippocampus or hypothalamus. It is concluded that morphine does not affect the neurosteroid modulatory effect on ifenprodil binding in the rat hippocampus or hypothalamus but does significantly affect both the expression of the NR2B subunit and the 3α5ßS modulatory effect on ifenprodil binding in the frontal cortex. It is suggested that the observed effect of long term morphine on the properties of NR2B in the frontal cortex may be associated with the mechanism underlying the development of opiate dependence.

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α1ß3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3ß-epimer epi-allopregnanolone, binds to the canonical ß3(+)-α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the ß3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

Kinetic analysis of voltage-dependent potentiation and block of the glycine alpha 3 receptor by a neuroactive steroid analogue.

We examined the actions of a carboxylated analogue of pregnanolone ((3alpha,5beta)-20-oxopregnane-3-carboxylic acid; 3alphaCOOH5betaP) on receptors composed of glycine receptor alpha3 subunits, expressed in Xenopus oocytes. This analogue both inhibits and potentiates this receptor; potentiation increases with more negative membrane potentials while block increases with less negative membrane potentials. We used a second analogue ((3alpha,5beta)-3-hydroxymethylpregnan-20-one; 3alphaCH(2)OH5betaP) to examine the mechanism for voltage-dependent potentiation. This analogue potentiates but does not block the glycine alpha3 receptor. Steady-state responses and current relaxations following voltage jumps support the idea that the voltage dependence of potentiation indirectly arises from a voltage dependence for channel activation by glycine, rather than an intrinsic voltage dependence for potentiation. Potentiation results from a slowing of the channel deactivation rate. In the absence of steroid, at a low [glycine] current relaxations after a voltage jump show two exponential components, with a weighted average time constant of approximately 425 ms (-50 mV, 22 degrees C). The rate for channel deactivation increases at more negative potentials (e-fold per 170 mV) whereas activation decreases (e-fold per 230 mV). The probability a channel is active at a high [glycine] is greater than 0.9. The addition of 10 microM 3alphaCH(2)OH5betaP decreases the deactivation rate by 6.3-fold (-50 mV). Voltage-dependent block by 3alphaCOOH5betaP is consistent with simple open-channel block, with voltage dependence reflecting interactions of the charge on the analogue with the electrical field. Block and unblock have equal but opposite dependence on membrane potential, and the charge on 3alphaCOOH5betaP senses approximately 70% of the membrane field at the blocking site. The apparent forward rate for block, however, is very slow (2 x 10(5) m(-1) s(-1)).

Plasticity of the alpha4betadelta GABAA receptor.

The GABAR [GABA(A) (gamma-aminobutyric acid type A) receptor], which mediates most inhibition in the brain, is regulated homoeostatically to maintain an optimal level of neuronal excitability. In particular, the alpha(4)betadelta subtype of the GABAR plays a pivotal role in this regulation. This receptor, which is expressed extrasynaptically on the dendrites, normally has low expression in the brain, but displays a remarkable degree of plasticity. It can also be a sensitive target for endogenous neurosteroids such as THP (3alpha-hydroxy-5[alpha]beta-pregnan-20-one (allo-pregnanolone); a neurosteroid and positive modulator of the GABAR), which is released during stress, although the effect of the steroid is polarity-dependent, such that it increases inward current, but decreases outward current, at alpha(4)beta(2)delta GABAR. Expression of alpha(4)beta(2)delta GABAR in CA1 hippocampus is also tightly regulated by fluctuating levels of neurosteroids, as seen at the onset of puberty. Declining levels of inhibition resulting from the decrease in THP at puberty are compensated for by an increase in alpha(4)betadelta GABAR along the apical dendrites of CA1 hippocampal pyramidal cells, which reduces neuronal excitability by decreasing the input resistance. However, excessive decrease of neuronal function is averted when THP levels rise, as would occur during stress, because this steroid decreases the outward GABAergic tonic current via inhibition of alpha(4)beta(2)delta GABAR, thereby restoring measures of neuronal excitability to pre-pubertal levels. Thus the homoeostatic regulation of alpha(4)betadelta GABAR expression plays an important role in maintaining ambient levels of neuronal excitability at puberty.