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Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epiallopregnanolone (3β-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 3β-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.

Synaptic GABAA receptors are alternately exposed to short pulses of a high, millimolar concentration of GABA and prolonged periods of low, micromolar concentration of the transmitter. Prior work has indicated that exposure to micromolar concentrations of GABA can both activate the postsynaptic receptors generating sustained low-amplitude current and desensitize the receptors, thereby reducing the peak amplitude of subsequent synaptic response. However, the precise relationship between tonic activation and reduction of peak response is not known. Here, we have measured the effect of prolonged exposure to GABA or the combination of GABA and the neurosteroid allopregnanolone, which was intended to desensitize a fraction of receptors, on a subsequent response to a high concentration of agonist in human α1β3γ2L receptors expressed in Xenopus oocytes. We show that the reduction in the peak amplitude of the post-exposure test response correlates with the open probability of the preceding desensitizing response. Curve fitting of the inhibitory relationship yielded an IC50 of 12.5 µM and a Hill coefficient of -1.61. The activation and desensitization data were mechanistically analyzed in the framework of a three-state Resting-Active-Desensitized model. Using the estimated affinity, efficacy, and desensitization parameters, we calculated the amount of desensitization that would accumulate during a long (2-minute) application of GABA or GABA plus allopregnanolone. The results indicate that accumulation of desensitization depends on the level of activity rather than agonist or potentiator concentration per se. We estimate that in the presence of 1 µM GABA, approximately 5% of α1β3γ2L receptors are functionally eliminated because of desensitization. SIGNIFICANCE STATEMENT: We present an analytical approach to quantify and predict the loss of activatable GABAA receptors due to desensitization in the presence of transmitter and the steroid allopregnanolone. The findings indicate that the peak amplitude of the synaptic response is influenced by ambient GABA and that changes in ambient concentrations of the transmitter and other GABAergic agents can modify tonically and phasically activated synaptic receptors in opposite directions.

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 5β-reduced steroids, sulfated or carboxylated steroids, and β-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 (3α5βP), 3α5βP sulfate, and β-estradiol. The site for 3α5βP 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.

This study examines how site-specific binding to the three identified neurosteroid binding sites in the α1β3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β3(+)–α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting receptor desensitization and the α1 subunit promoting ligand-specific effects. 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 site-specific and general 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.

Background The mechanisms underlying the role of T-type calcium channels (T-channels) in thalamocortical excitability and oscillations in vivo during neurosteroid-induced hypnosis are largely unknown. Methods We used patch-clamp electrophysiological recordings from acute brain slices ex vivo, recordings of local field potentials (LFPs) from the central medial thalamic nucleus in vivo, and wild-type (WT) and Cav3.1 knock-out mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH). Results Patch-clamp recordings showed that 3β-OH inhibited isolated T-currents but had no effect on phasic or tonic γ-aminobutyric acid A currents. Also in acute brain slices, 3β-OH inhibited the spike firing mode more profoundly in WT than in Cav3.1 knockout mice. Furthermore, 3β-OH significantly hyperpolarised neurones, reduced the amplitudes of low threshold spikes, and diminished rebound burst firing only in WT mice. We found that 80 mg kg−1 i.p. injections of 3β-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3β-OH (20 mg kg−1 i.p.) accelerated induction of hypnosis by isoflurane only in WT mice, but had similar effects on the maintenance of isoflurane-induced hypnosis in both WT and Cav3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3β-OH increased δ, θ, α, and β oscillations in WT mice in comparison with Cav3.1 knock-out mice. Conclusions The Cav3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.

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 β-α subunit interface (site I) and within the α (site II) and β subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1β2γ2L 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.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action., Novel neurosteroid analogue photolabeling reagents identify three specific neurosteroid binding sites on α1β3 GABAA receptors, showing that a site between the α and β subunits, as well as a site within the α-subunit, contribute to neurosteroid-mediated enhancement of GABAA currents., Author summary Neurosteroids are cholesterol metabolites produced by neurons and glial cells that participate in central nervous system (CNS) development, regulate neuronal excitability, and modulate complex behaviors such as mood. Exogenously administered neurosteroid analogues are effective sedative hypnotics and are being developed as antidepressants and anticonvulsants. Gamma amino-butyric acid Type A (GABAA) receptors, the principal ionotropic inhibitory neurotransmitter receptors in the brain, are the primary functional target of neurosteroids. Understanding the molecular details of neurosteroid interactions with GABAA receptors is critical to understanding their mechanism of action and developing specific and effective therapeutic agents. In the current study, we developed a suite of neurosteroid analogue affinity labeling reagents, which we used to identify three distinct binding sites on GABAA receptors and to determine the orientation of neurosteroid binding in each site. Electrophysiological studies performed on receptors with mutations designed to disrupt the identified binding sites showed that two of the three sites contribute to neurosteroid modulation of GABAA currents. The distinct patterns of neurosteroid affinity, binding orientation, and effect provide the potential for the development of isoform-specific agonists, partial agonists, and antagonists with targeted therapeutic effects.

Many GABAergic drugs are in clinical use as anesthetics, sedatives, or anxiolytics. We have investigated the actions of the combinations of the neuroactive steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with the intravenous anesthetic propofol or the benzodiazepine diazepam. The goal of the study was to determine whether coapplication of alfaxalone reduces the effective doses and concentrations of propofol and diazepam. Behavioral effects of alfaxalone, propofol, diazepam, and the combinations of the drugs were evaluated during a 30-min activity test in mice. Functional effects of the individual drugs and drug combinations were tested by measuring the decay times of spontaneous inhibitory postsynaptic currents in rat hippocampal neurons, and peak current responses from heterologously expressed concatemeric α1β2γ2L GABAA receptors. Co-administration of alfaxalone increased the sedative actions of propofol and diazepam in mice. The combination of alfaxalone with propofol or diazepam increased the decay times of sIPSCs and shifted the concentration-response relationships for GABA-activated receptors to lower transmitter concentrations. We infer that alfaxalone acts as a co-agonist to enhance the GABAergic effects of propofol and diazepam. We propose that co-administration of alfaxalone, and possibly other neuroactive steroids, can be employed to reduce dosage requirements for propofol and diazepam.

Background and Purpose Neurosteroids potentiate responses of the GABAA receptor to the endogenous agonist GABA. Here, we examined the ability of neurosteroids to potentiate responses to the allosteric activators etomidate, pentobarbital and propofol. Experimental Approach Electrophysiological assays were conducted on rat α1β2γ2L GABAA receptors expressed in HEK 293 cells. The sedative activity of etomidate was studied in Xenopus tadpoles and mice. Effects of neurosteroids on etomidate-elicited inhibition of cortisol synthesis were determined in human adrenocortical cells. Key Results The neurosteroid 5β-pregnan-3α-ol-20-one (3α5βP) potentiated activation of GABAA receptors by GABA and allosteric activators. Co-application of 1 μM 3α5βP induced a leftward shift (almost 100-fold) of the whole-cell macroscopic concentration–response relationship for gating by etomidate. Co-application of 100 nM 3α5βP reduced the EC50 for potentiation by etomidate of currents elicited by 0.5 μM GABA by about three-fold. In vivo, 3α5βP (1mg kg-1) reduced the dose of etomidate required to produce loss of righting in mice (ED50) by almost 10-fold. In tadpoles, the presence of 50 or 100 nM 3α5βP shifted the EC50 for loss of righting about three- or ten-fold respectively. Exposure to 3α5βP did not influence inhibition of cortisol synthesis by etomidate. Conclusions and Implications Potentiating neurosteroids act similarly on orthosterically and allosterically activated GABAA receptors. Co-application of neurosteroids with etomidate can significantly reduce dosage requirements for the anaesthetic, and is a potentially beneficial combination to reduce undesired side effects.

Neurosteroids positively modulate GABA-A receptor (GABA(A)R) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABA(A)R chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

A model of the alignment of neurosteroids and ent-neurosteroids at the same binding site on γ-aminobutyric acid type A (GABAA) receptors was evaluated for its ability to identify the structural features in ent-neurosteroids that enhance their activity as positive allosteric modulators of this receptor. Structural features that were identified included: (1) a ketone group at position C-16, (2) an axial 4α-OMe group, and (3) a C-18 methyl group. Two ent-steroids were identified that were more potent than the anesthetic steroid alphaxalone in their threshold for and duration of loss of the righting reflex in mice. In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50 values similar to those found for allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anesthetic agents and as drugs to treat brain disorders that are ameliorated by positive allosteric modulators of GABAA receptor function.

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors.

Alphaxalone, a neuroactive steroid containing a 17β-acetyl group, has potent anesthetic activity in humans. This pharmacological activity is attributed to this steroid's enhancement of γ-amino butyric acid-mediated chloride currents at γ-amino butyric acid type A receptors. The conversion of alphaxalone into Δ(16)-alphaxalone produces an analogue that lacks anesthetic activity in humans and that has greatly diminished receptor actions. By contrast, the corresponding 17β-carbonitrile analogue of alphaxalone and the Δ(16)-17-carbonitrile analogue both have potent anesthetic and receptor actions. The differential effect of the Δ(16)-double bond on the actions of alphaxalone and the 17β-carbonitrile analogue is accounted for by a differential effect on the orientation of the 17-acetyl and 17-carbonitrile substituents.

Endogenous neurosteroids and synthetic neuroactive steroid analogs are among the most potent and efficacious potentiators of the mammalian GABA-A receptor. The compounds interact with one or more sites on the receptor leading to an increase in the channel open probability through a set of changes in the open and closed time distributions. The endogenous neurosteroid allopregnanolone potentiates the α1β2γ2L GABA-A receptor by enhancing the mean duration and prevalence of the longest-lived open time component and by reducing the prevalence of the longest-lived intracluster closed time component. Thus the channel mean open time is increased and the mean closed time duration is decreased, resulting in potentiation of channel function. Some of the other previously characterized neurosteroids and steroid analogs act through similar mechanisms while others affect a subset of these parameters. The steroids modulate the GABA-A receptor through interactions with the membrane-spanning region of the receptor. However, the number of binding sites that mediate the actions of steroids is unclear. We discuss data supporting the notions of a single site vs. multiple sites mediating the potentiating actions of steroids.

We have shown that fluorescent, 7-nitro-2,1,3-benzoxadiazol-4-yl amino (NBD)-conjugated neurosteroid analogs photopotentiate GABA(A) receptor function. These compounds seem to photosensitize a modification of receptor function, resulting in long-lived increases in responses to exogenous or synaptic GABA. Here we extend this work to examine the effectiveness of different fluorophore positions, conjugations, steroid structures, and fluorophores. Our results are generally in agreement with the idea that steroids with activity at GABA(A) receptors are the most potent photopotentiators. In particular, we find that an unnatural enantiomer of an effective photopotentiating steroid is relatively weak, excluding the idea that membrane solubility alone, which is identical for enantiomer pairs, is solely responsible for potent photopotentiation. Furthermore, there is a significant correlation between baseline GABA(A) receptor activity and photopotentiation. Curiously, both sulfated steroids, which bind a presumed external neurosteroid antagonist site, and hydroxysteroids, which bind an independent site, are effective. We also find that a rhodamine dye conjugated to a 5beta-reduced 3alpha-hydroxy steroid is a particularly potent and effective photopotentiator, with minimal baseline receptor activity up to 10 muM. Steroid conjugated fluorescein and Alexa Fluor 546 also supported photopotentiation, although the Alexa Fluor conjugate was weaker and required 10-fold higher concentration to achieve similar potentiation to the best NBD and rhodamine conjugates. Filling cells with steroid-conjugated or free fluorophores via whole-cell patch pipette did not support photopotentiation. FM1-43, another membrane-targeted, structurally unrelated fluorophore, also produced photopotentiation at micromolar concentrations. We conclude that further optimization of fluorophore and carrier could produce an effective, selective, light-sensitive GABA(A) receptor modulator.

Background and purpose: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABAA receptor. Experimental approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant α1β2γ2L GABAA receptors. Key results: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat α1β2γ2L GABAA receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues α1Q241 and α1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3α,5α)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABAA receptors activated by very high concentrations (1–10 μM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. Conclusions and implications: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABAA receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABAA receptor modulators. British Journal of Pharmacology (2008) 153, 598–608; doi:10.1038/sj.bjp.0707597; published online 26 November 2007

Allopregnanolone (1) and pregnanolone (2), steroids containing a 17beta-acetyl group, are potent enhancers of GABA (gamma-aminobutyric acid) action at GABAA receptors. Their effects are enantioselective with the non-naturally occurring enantiomers (ent-1 and ent-2) being less potent. Androsterone (3) and etiocholanolone (4), steroids with a C-17 carbonyl group, are weak enhancers of GABA action at GABAA receptors. Unexpectedly, their enantiomers (ent-3 and ent-4) have been found to have enhanced, not diminished, activity at GABAA receptors. Furthermore, the C-17 spiro-epoxide analogues (ent-5 and ent-6) of ent-3 and ent-4, respectively, have activities comparable to those of steroids 1 and 2. The results indicate that some ent-steroids are potent modulators of GABAA receptors and might have clinical potential as GABAergic drugs of the future.

Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the beta configuration at the C2 position of (3alpha,5alpha)-3-hydroxypregnan-20-one (allopregnanolone, 3alpha5alphaP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.

We have examined alpha1beta2gamma2L GABAA receptor modulation by the endogenous steroids allopregnanolone (3alpha5alphaP), pregnenolone sulfate, and beta-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3alpha5alphaP but not that by pregnenolone sulfate or beta-estradiol. One of the three kinetic effects evident in channel potentiation by 3alpha5alphaP, prolongation of the longest-lived open time component (OT3), was affected by ethanol with the midpoint of its dose-response curve moved to lower steroid concentrations by 2 orders of magnitude without significantly affecting the maximal effect. Manipulations designed to affect the ability of 3alpha5alphaP to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the gamma2 subunit, which reduces the ability of 3alpha5alphaP to prolong OT3, also reduces the interaction between ethanol and 3alpha5alphaP. And the presence of the competitive steroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3alpha5alphaP on the GABAA receptor. Together, the findings suggest that steroid interactions with the classic steroid binding site underlie the effect seen in the presence of ethanol, and that ethanol acts by increasing the affinity of 3alpha5alphaP for the site. Tadpole behavioral assays showed that the presence of 3alpha5alphaP at a concentration ineffective at causing changes in tadpole behavior shifted the ethanol dose-response curve for loss of righting reflex to lower concentrations and that this effect was neutralized by coapplication of 17-PA with 3alpha5alphaP.

Benz[f]indenes are tricyclic compounds with a linear 6-6-5 fused carbocyclic ring system. When properly substituted, benz[f]indenes can satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids that modulate gamma-aminobutyric acidA (GABAA) receptor function. Thus, the benz[f]indene ring system provides an opportunity to extend the previously well-studied GABAA receptor structure-activity relationships (SAR) of neuroactive steroids to a different ring system. Depending on whether the stereochemistry of the 6-6-5 ring fusions are trans-trans or cis-trans, either planar or nonplanar benz[f]indenes are obtained. We found that the planar trans-trans benz[f]indenes are active, but less active than the steroids they were designed to mimic, whereas the nonplanar cis-trans compounds have little, if any, activity. The results provide new insight into the importance of the steroid framework for the actions of neuroactive steroids at GABAA receptors.

The planar 5α-reduced steroid (3α,5α)-3-hydroxypregnan-20-one and the nonplanar 5β-reduced steroid (3α,5β)-3-hydroxypregnan-20-one act at GABAA receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABAA receptors have not been determined. To determine how structural modifications at the steroid C-6 and C-7 positions effect the actions of these anesthetic steroids, an axial or equatorial methyl group was introduced at these positions. The analogues were evaluated (1) in [35S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat α1β2γ2L GABAA receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5α- and 5β-reduced series of compounds were strikingly similar. In both series, a 6β-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6α-, 7β- or 7α-Me substituent resulted in reduc...

The effects of neuroactive steroids on the function of GABAA receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with α1 β2 γ2L subunits. Activity was elicited with a half-maximal (50 μm) concentration of GABA. Two steroids were studied in detail: ACN ((3α,5α,17β)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3α,5β,17β)-3-hydroxy-18-norandrostane-17-carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration-dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nm ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nm ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nm B285) but increased the duration only at concentrations close to 10 μm. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nm ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation-related closed state selectively, with comparable IC50 concentrations (about 40 nm ACN, 20 nm B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 μm ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the γ2 subunit, but not the α1 or β2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABAA receptor.

Neuroactive steroids modulate the function of gamma-aminobutyric acid type A (GABA(A)) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3alpha,5beta)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA(A) receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA(A) receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA(A) receptor function and anesthesia. Photolabeling of 35-kDa protein with [(3)H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA(A) receptor function as evidenced by radioligand ([(35)S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA(A) receptor function normally in VDAC-2-deficient fibroblasts transfected with alpha(1)beta(2)gamma(2) GABA(A) receptor subunits. Finally, the neuroactive steroid pregnanolone [(3alpha,5beta)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1- and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA(A) receptor modulation or anesthesia.

The hydrogen-bond-acceptor properties of the carbonyl moiety in the 17beta-acetyl group on the D-ring of the anesthetic steroids (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregan-20-one form an important part of the anesthetic steroid pharmacophore. 13,24-Cyclo-18,21-dinorcholanes containing a ketone or conjugated ketone group at C-20, C-22, C-23, or C-24 were prepared as conformationally constrained analogues of these anesthetic steroids and were used to probe for alternate locations for the D-ring hydrogen-bond-accepting carbonyl group. The analogues were evaluated (1). in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2). in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3). as tadpole anesthetics. In the binding assay, the relative order of potencies for the analogues in the 5alpha- and 5beta-series is identical. For the ketones, the order is 24-oneor= 23-one20-one22-one. Likewise, for the enones, the order is delta(22)-24-onedelta(20(22))-23-onedelta(22)-20-onedelta(23)-22-one. Similar relative orders of potencies are also found in the other two bioassays. The activities of the 24-one and delta(22)-24-one compounds were expected to be very low, because the carbonyl group in these compounds is located over the steroid C-ring and oriented toward C-8. Instead, these compounds have the highest activities in their respective series, with the delta(22)-24-one compounds having activities comparable to those of the reference anesthetic steroids. The electrophysiology results obtained with the 24-oxo-cyclosteroids suggest that rat alpha(1)beta(2)gamma(2L) GABA(A) receptors contain more than one donor for the hydrogen-bond-acceptor group of anesthetic steroids. The family of cyclosteroids should be useful for future structure-activity relationship studies of steroid modulation of other GABA(A) receptor subtypes.

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.

While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified.This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors.Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific.CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

The synthesis of (3β,5β,8α,9β,10α,13α,14β,17α)-3-hydroxypregnan-20-one 2, (3β,5β,8α,9β,10α,13α,14β,17α)-3-hydroxyandrostane-17-carbonitrile 4,† [3R-(3α,3aα,5aβ,7α,9aα,9bβ)]-1-[dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]inden-3-yl]ethanone 6 and [3R-(3α,3aα,5aβ,7α,9aα,9bβ)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile 8 is reported.‡ Steroids 2 and 4 have been used previously to investigate the enantioselectivity of steroid action on GABAA receptor function and to correlate steroid action at this receptor with the anesthetic actions of the compounds. The enantioselective actions of benz[e]indenes 6 and 8 have been evaluated for the same reasons in this study. Similar correlations between the enantioselective effects of both classes of compounds on GABAA receptor function and anesthetic potency in tadpoles have been observed.

The enantiomer pair androsterone and ent-androsterone are positive allosteric modulators of γ-aminobutyric acid (GABA) type A receptors. Each enantiomer was shown to bind at the same receptor site. Binding orientations of the enantiomers at this site were deduced using enantiomer pairs containing OBn substituents at either C-7 or C-11. 11β-OBn-substituted steroids and 7α-OBn-substituted ent-steroids potently displace [(35)S]-tert-butylbicyclophosphorothionate, augment GABA currents, and anesthetize tadpoles. In contrast, 7β-OBn-substituted steroids and 11α-OBn-substituted ent-steroids have diminished actions. The results suggest that the binding orientations of the active analogues are inverted relative to each other with the 7α- and 11β-substituents similarly located on the edges of the molecules not in contact with the receptor surface. Analogue potentiation of the GABA current was abrogated by an α(1) subunit Q241L mutation, indicating that the active analogues act at the same sites in α(1)β(2)γ(2L) receptors previously associated with positive neurosteroid modulation.

This study addresses the hypothesis that the lack of anesthetic activity for (3α,5α)-3-hydroxypregn-16-ene-11,20-dione (Δ(16)-alphaxalone) is explained by the steroid Δ(16) double bond constraining the steroid 20-carbonyl group to a position that prevents it from favorably interacting with γ-aminobutyric acid type A (GABA(A)) receptors. A series of Δ(16) and Δ(17(20)) analogues of Δ(16)-alphaxalone was prepared to evaluate this hypothesis in binding, electrophysiological, and tadpole anesthesia experiments. The results obtained failed to support the hypothesis. Instead, the results indicate that it is the presence of the C-21 methyl group in Δ(16)-alphaxalone, not the location of the constrained C-20 carbonyl group, that prevents Δ(16)-alphaxalone from interacting strongly with the GABA(A) receptor and having anesthetic activity. Consistent with this conclusion, a Δ(17(20)) analogue of Δ(16)-alphaxalone without a C-21 methyl group was found to be very similar to the anesthetic steroid (3α,5α)-3-hydroxypregnane-11,20-dione (alphaxalone) with regard to time of onset and rate of recovery from anesthesia when administered to mice by tail vein injection.

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.

Modern views of anesthetic neurosteroid interaction with the GABA(A) receptor conceptualize steroid ligands interacting with a protein binding site on the receptor. It has generally been assumed that the steroid interaction/binding site is contained in an extracellular domain of the receptor, and that steroid interactions are of high potency, evidenced by the low aqueous ligand concentrations required to achieve potentiation of channel function. We have been considering implications of the observations that steroids are quite lipophilic and that recently identified putative steroid binding sites are in transmembrane domains of the receptor. Accordingly, we expect that both the effective plasma membrane steroid concentration and steroid pharmacophore properties will contribute to steady-state potency and to the lifetime of steroid actions following removal of free aqueous steroid. Here we review our recent studies that address the evidence that membrane partitioning and intracellular accumulation are non-specific contributors to the effects of anesthetic steroids at GABA(A) receptors. We compare and contrast the profile of anesthetic steroids with that of sulfated steroids that negatively regulate GABA(A) receptor function. These studies give rise to the view that the inherent affinity of anesthetic steroid for GABA(A) receptors is very low; low effective aqueous concentrations are accounted for by lipid partitioning. This yields a very different picture of the interaction of neurosteroids with the GABA(A) receptor than that of steroid interactions with classical intracellular steroid receptors, which exhibit inherently high affinity. These considerations have practical implications for actions of endogenous neurosteroids. Lipophilicity will tend to promote autocrine actions of neurosteroids at GABA(A) receptors within cells that synthesize neurosteroids, and lipophilic retention will limit intercellular diffusion from the source of steroid synthesis. Lipophilicity and steroid access to the receptor binding sites also must be considerations in drug design if drugs are to effectively reach the target GABA(A) receptor site.

Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).

Neuroactive steroids have some of their most potent actions by augmenting the function of GABA(A) receptors. Endogenous steroid actions on GABA(A) receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABA(A) receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel.

Although neurosteroids have rapid effects on GABA(A) receptors, study of steroid actions at GABA receptors has been hampered by a lack of pharmacological antagonists. In this study, we report the synthesis and characterization of a steroid analog, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA), that selectively antagonized neurosteroid potentiation of GABA responses. We examined 17PA using the alpha1beta2gamma2 subunit combination expressed in Xenopus laevis oocytes. 17PA had little or no effect on baseline GABA responses but antagonized both the response augmentation and the direct gating of GABA receptors by 5alpha-reduced potentiating steroids. The effect was selective for 5alpha-reduced potentiating steroids; 5beta-reduced potentiators were only weakly affected. Likewise, 17PA did not affect barbiturate and benzodiazepine potentiation. 17PA acted primarily by shifting the concentration response for steroid potentiation to the right, suggesting the possibility of a competitive component to the antagonism. 17PA also antagonized 5alpha-reduced steroid potentiation and gating in hippocampal neurons and inhibited anesthetic actions in X. laevis tadpoles. Analogous to benzodiazepine site antagonists, the development of neurosteroid antagonists may help clarify the role of GABA-potentiating neurosteroids in health and disease.

GABAergic neurotransmission can be both positively and negatively modulated by steroids. The steroid effects are thought to be mediated by binding of steroids to specific sites on GABAA receptors. It appears that the receptor sites for positive and negative modulatory steroids are different. Thus far, the location and number of binding sites for steroids on these receptors have not been established. In this brief review, we concentrate largely on results from our own structure–activity studies. Novel analogues have been studied to further delineate the structural features required for compounds to modulate receptor function via steroid binding sites. Non-naturally occurring enantiomers of both positive and negative modulators have been studied to provide further evidence for the existence of specific steroid binding sites on the receptors.

Steroids, in addition to regulating gene expression, directly affect a variety of ion channels. We examined the action of steroids on human embryonic kidney 293 cells stably transfected to express rat alpha4beta2 neuronal nicotinic receptors. Each steroid that was tested inhibited acetylcholine responses from these receptors, with slow kinetics requiring seconds for block to develop and recover. The action of one steroid [3alpha,5alpha, 17beta-3-hydroxyandrostane-17-carbonitrile (ACN)] was studied in detail. Block showed enantioselectivity, with an IC(50) value of 1.5 microM for ACN and 4.5 microM for the enantiomer. Inhibition curves had Hill slopes larger than 1, indicating more than one binding site per receptor. Block did not require intracellular compounds containing high-energy phosphate bonds and was not affected by analogs of GTP, suggesting that the mechanism does not require the activation of second messengers. Block did not appear to be strongly selective between open and closed channel states or to involve changes in desensitization. A comparison of different steroids showed that a beta-orientation of groups at the 17 position produced more block than alpha-orientated diastereomers. The stereochemistry at the 3 and 5 positions was less influential for block of alpha4beta2 nicotinic receptors, despite its importance for potentiation of gamma-aminobutyric acid(A) receptors. The ability of steroids to block neuronal nicotinic receptors correlated with their ability to produce anesthesia in Xenopus tadpoles, but the concentrations required for inhibition are generally greater. Similarly, the concentrations of endogenous neurosteroids required to inhibit receptors are larger than estimates of brain concentrations.