Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

Comparison of Behavioral Effects of GABAergic Low- and High-Efficacy Neuroactive Steroids in the Zebrafish Larvae Assay.

Activation of the GABAA receptor is associated with numerous behavioral end points ranging from anxiolysis to deep anesthesia. The specific behavioral effect of a GABAergic compound is considered to correlate with the degree of its functional effect on the receptor. Here, we tested the hypothesis that a low-efficacy allosteric potentiator of the GABAA receptor may act, due to a ceiling effect, as a sedative with reduced and limited action. We synthesized a derivative, named (3α,5ß)-20-methyl-pregnane-3,20-diol (KK-235), of the GABAergic neurosteroid 5ß-pregnane-3α,20α-diol. Using electrophysiology, we showed that KK-235 is a low-efficacy potentiator of the synaptic-type α1ß2γ2L GABAA receptor. In the zebrafish larvae behavioral assay, KK-235 was found to only partially block the inverted photomotor response (PMR) and to weakly reduce swimming behavior, whereas the high-efficacy GABAergic steroid (3α,5α,17ß)-3-hydroxyandrostane-17-carbonitrile (ACN) fully blocked PMR and spontaneous swimming. Coapplication of KK-235 reduced the potentiating effect of ACN in an electrophysiological assay and dampened its sedative effect in behavioral experiments. We propose that low-efficacy GABAergic potentiators may be useful as sedatives with limited action.

UNC-49 is a redox-sensitive GABAA receptor that regulates the mitochondrial unfolded protein response cell nonautonomously.

The γ-aminobutyric acid-mediated (GABAergic) system participates in many aspects of organismal physiology and disease, including proteostasis, neuronal dysfunction, and life-span extension. Many of these phenotypes are also regulated by reactive oxygen species (ROS), but the redox mechanisms linking the GABAergic system to these phenotypes are not well defined. Here, we report that GABAergic redox signaling cell nonautonomously activates many stress response pathways in Caenorhabditis elegans and enhances vulnerability to proteostasis disease in the absence of oxidative stress. Cell nonautonomous redox activation of the mitochondrial unfolded protein response (mitoUPR) proteostasis network requires UNC-49, a GABAA receptor that we show is activated by hydrogen peroxide. MitoUPR induction by a spinocerebellar ataxia type 3 (SCA3) C. elegans neurodegenerative disease model was similarly dependent on UNC-49 in C. elegans. These results demonstrate a multi-tissue paradigm for redox signaling in the GABAergic system that is transduced via a GABAA receptor to function in cell nonautonomous regulation of health, proteostasis, and disease.

Potentiation of the GABAAR reveals variable energetic contributions by etiocholanolone and propofol.

The properties of a potentiator are typically evaluated by measuring its ability to enhance the magnitude of the control response. Analysis of the ability of drugs to potentiate responses from receptor channels takes place in the context of particular models to extract parameters for functional effects. In the often-used coagonist model, the agonist generating control activity and the potentiator enhancing the control activity make additive energetic contributions to stabilize the active state of the receptor. The energetic contributions are fixed and, once known, enable calculation of predicted receptor behavior at any concentration combination of agonist and potentiator. Here, we have examined the applicability of the coagonist model by measuring the relationship between the magnitude of receptor potentiation and the level of background activity. Ternary αßγ GABAA receptors were activated by GABA or the allosteric agonist propofol, or by a gain-of-function mutation, and etiocholanolone- or propofol-mediated potentiation of peak responses was measured. We show that the free energy change contributed by the modulators etiocholanolone or propofol is reduced at higher levels of control activity, thereby being in disagreement with basic principles of the coagonist model. Possible mechanisms underlying this discrepancy are discussed.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABAA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABAA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABAA and the nicotinic α7 receptor modulator (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary αßγ GABAA receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-N-phenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (ΔG ∼ -1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the α1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: We have synthesized and characterized the actions of novel acrylamide-derived compounds on the mammalian GABAA receptor. We show that the compounds have concurrent potentiating effects mediated by the classic anesthetic binding sites, and inhibitory actions that bear mechanistic resemblance to but do not share binding sites with, the inhibitory steroid pregnenolone sulfate.

Mutational Analysis of Anesthetic Binding Sites and Their Effects on GABAA Receptor Activation and Modulation by Positive Allosteric Modulators of the α7 Nicotinic Receptor.

The positive allosteric modulators (PAMs) of the α7 nicotinic receptor N-(5-Cl-2-hydroxyphenyl)-N'-[2-Cl-5-(trifluoromethyl)phenyl]-urea (NS-1738) and (E)-3-(furan-2-yl)-N-(p-tolyl)-acrylamide (PAM-2) potentiate the α1ß2γ2L GABAA receptor through interactions with the classic anesthetic binding sites located at intersubunit interfaces in the transmembrane domain of the receptor. In the present study, we employed mutational analysis to investigate in detail the involvement and contributions made by the individual intersubunit interfaces to receptor modulation by NS-1738 and PAM-2. We show that mutations to each of the anesthetic-binding intersubunit interfaces (ß+/α-, α+/ß-, and γ+/ß-), as well as the orphan α+/γ- interface, modify receptor potentiation by NS-1738 and PAM-2. Furthermore, mutations to any single interface can fully abolish potentiation by the α7-PAMs. The findings are discussed in the context of energetic additivity and interactions between the individual binding sites.

The Mechanism of Enantioselective Neurosteroid Actions on GABAA Receptors.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that ent-ALLO potentiates GABA-elicited currents in α1ß3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the ß3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the ß3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or ß3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the ß3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.

Modulation of the mammalian GABAA receptor by type I and type II positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

BACKGROUND AND PURPOSE: Positive allosteric modulators of the α7 nicotinic acetylcholine (nACh) receptor (α7-PAMs) possess promnesic and procognitive properties and have potential in the treatment of cognitive and psychiatric disorders including Alzheimer's disease and schizophrenia. Behavioural studies in rodents have indicated that α7-PAMs can also produce antinociceptive and anxiolytic effects that may be associated with positive modulation of the GABAA receptor. The overall goal of this study was to investigate the modulatory actions of selected α7-PAMs on the GABAA receptor. EXPERIMENTAL APPROACH: We employed a combination of cell fluorescence imaging, electrophysiology, functional competition and site-directed mutagenesis to investigate the functional and structural mechanisms of modulation of the GABAA receptor by three representative α7-PAMs. KEY RESULTS: We show that the α7-PAMs at micromolar concentrations enhance the apparent affinity of the GABAA receptor for the transmitter and potentiate current responses from the receptor. The compounds were equi-effective at binary αß and ternary αßγ GABAA receptors. Functional competition and site-directed mutagenesis indicate that the α7-PAMs bind to the classic anaesthetic binding sites in the transmembrane region in the intersubunit interfaces, which results in stabilization of the active state of the receptor. CONCLUSION AND IMPLICATIONS: We conclude that the tested α7-PAMs are micromolar-affinity, intermediate- to low-efficacy allosteric potentiators of the mammalian αßγ GABAA receptor. Given the similarities in the in vitro sensitivities of the α7 nACh and α1ß2γ2L GABAA receptors to α7-PAMs, we propose that doses used to produce nACh receptor-mediated behavioural effects in vivo are likely to modulate GABAA receptor function.

Activation of the Rat α1ß2ε GABAA Receptor by Orthosteric and Allosteric Agonists.

GABAA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABAA subunits cloned to date. GABAA receptors containing the ε subunit exhibit a significant degree of constitutive activity and have been suggested to be unresponsive to allosteric agents. In this study, we have characterized the functional properties of the rat α1ß2ε GABAA receptor. We confirm that the α1ß2ε receptor exhibits a higher level of constitutive activity than typical of GABAA receptors and show that it is inefficaciously activated by the transmitter and the allosteric agonists propofol, pentobarbital, and allopregnanolone. Manipulations intended to alter ε subunit expression and receptor stoichiometry were largely without effect on receptor properties including sensitivity to GABA and allosteric agonists. Surprisingly, amino acid substitutions at the conserved 9' and 6' positions in the second transmembrane (TM2) domain in the ε subunit did not elicit the expected functional effects of increased constitutive activity and resistance to the channel blocker picrotoxin, respectively. We tested the accessibility of TM2 residues mutated to cysteine using the cysteine-modifying reagent 4-(hydroxymercuri)benzoic acid and found a unique pattern of water-accessible residues in the ε subunit.

(+)-Catharanthine potentiates the GABAA receptor by binding to a transmembrane site at the ß(+)/α(-) interface near the TM2-TM3 loop.

(+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABAAR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABAAR subtype selectivity and location of the putative binding site for (+)-catharanthine using electrophysiological, site-directed mutagenesis, functional competition, and molecular docking experiments. Electrophysiological and in silico experiments showed that (+)-catharanthine potentiates the responses to low, subsaturating GABA at ß2/3-containing GABAARs 2.4-3.5 times more efficaciously than at ß1-containing GABAARs. The activity of (+)-catharanthine is reduced by the ß2(N265S) mutation that decreases GABAAR potentiation by loreclezole, but not by the ß3(M286C) or α1(Q241L) mutations that reduce receptor potentiation by R(+)-etomidate or neurosteroids, respectively. Competitive functional experiments indicated that the binding site for (+)-catharanthine overlaps that for loreclezole, but not those for R(+)-etomidate or potentiating neurosteroids. Molecular docking experiments suggested that (+)-catharanthine binds at the ß(+)/α(-) intersubunit interface near the TM2-TM3 loop, where it forms H-bonds with ß2-D282 (TM3), ß2-K279 (TM2-TM3 loop), and ß2-N265 and ß2-R269 (TM2). Site-directed mutagenesis experiments supported the in silico results, demonstrating that the K279A and D282A substitutions, that lead to a loss of H-bonding ability of the mutated residue, and the N265S mutation, impair the gating efficacy of (+)-catharanthine. We infer that (+)-catharanthine potentiates the GABAAR through several H-bond interactions with a binding site located in the ß(+)/α(-) interface in the transmembrane domain, near the TM2-TM3 loop, where it overlaps with loreclezole binding site.

The Sulfated Steroids Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate Inhibit the α1ß3γ2L GABAA Receptor by Stabilizing a Novel Nonconducting State.

The GABAA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α1ß3γ2L GABAA receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABAA receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state.

Perspective on the Relationship between GABAA Receptor Activity and the Apparent Potency of an Inhibitor.

BACKGROUND: In electrophysiological experiments, inhibition of a receptor-channel, such as the GABAA receptor, is measured by co-applying an agonist producing a predefined control response with an inhibitor to calculate the fraction of the control response remaining in the presence of the inhibitor. The properties of the inhibitor are determined by fitting the inhibition concentration- response relationship to the Hill equation to estimate the midpoint (IC50) of the inhibition curve Objective: We sought to estimate sensitivity of the fitted IC50 to the level of activity of the control response Methods: The inhibition concentration-response relationships were calculated for models with distinct mechanisms of inhibition. In Model I, the inhibitor acts allosterically to stabilize the resting state of the receptor. In Model II, the inhibitor competes with the agonist for a shared binding site. In Model III, the inhibitor stabilizes the desensitized state. RESULTS: The simulations indicate that the fitted IC50 of the inhibition curve is sensitive to the degree of activity of the control response. In Models I and II, the IC50 of inhibition was increased as the probability of being in the active state (PA) of the control response increased. In Model III, the IC50 of inhibition was reduced at higher PA. CONCLUSION: We infer that the apparent potency of an inhibitor depends on the PA of the control response. While the calculations were carried out using the activation and inhibition properties that are representative of the GABAA receptor, the principles and conclusions apply to a wide variety of receptor- channels.

Activation of the α1ß2γ2L GABAA Receptor by Physiological Agonists.

The Cl- permeable GABAA receptor is a major contributor to cellular inhibition in the brain. The receptor is normally activated by synaptically-released or ambient GABA but is sensitive to a number of physiological compounds such as ß-alanine, taurine, and neurosteroids that, to various degrees, activate the receptor and modulate responses either to the transmitter or to each other. Here, we describe α1ß2γ2L GABAA receptor activation and modulation by combinations of orthosteric and allosteric activators. The overall goal was to gain insight into how changes in the levels of endogenous agonists modulate receptor activity and influence cellular inhibition. Experimental observations and simulations are described in the framework of a cyclic concerted transition model. We also provide general analytical solutions for the analysis of electrophysiological data collected in the presence of combinations of active compounds.

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.

Enhancement of Muscimol Binding and Gating by Allosteric Modulators of the GABAA Receptor: Relating Occupancy to State Functions.

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria and a potent orthosteric agonist of the GABAA receptor. The binding of [3H]muscimol has been used to evaluate the distribution of GABAA receptors in the brain, and studies of modulation of [3H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABAA receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant α1ß3 GABAA receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [3H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. SIGNIFICANCE STATEMENT: The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [3H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate.

Reduced Activation of the Synaptic-Type GABAA Receptor Following Prolonged Exposure to Low Concentrations of Agonists: Relationship between Tonic Activity and Desensitization.

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 α1ß3γ2L 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 α1ß3γ2L 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.

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.

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.

Steady-state activation of the high-affinity isoform of the α4ß2δ GABAA receptor.

Activation of GABAA receptors consisting of α4, ß2 (or ß3), and δ subunits is a major contributor to tonic inhibition in several brain regions. The goal of this study was to analyze the function of the α4ß2δ receptor in the presence of GABA and other endogenous and clinical activators and modulators under steady-state conditions. We show that the receptor has a high constitutive open probability (~0.1), but is only weakly activated by GABA that has a maximal peak open probability (POpen,peak) of 0.4, taurine (maximal POpen,peak = 0.4), or the endogenous steroid allopregnanolone (maximal POpen,peak = 0.2). The intravenous anesthetic propofol is a full agonist (maximal POpen,peak = 0.99). Analysis of currents using a cyclic three-state Resting-Active-Desensitized model indicates that the maximal steady-state open probability of the α4ß2δ receptor is ~0.45. Steady-state open probability in the presence of combinations of GABA, taurine, propofol, allopregnanolone and/or the inhibitory steroid pregnenolone sulfate closely matched predicted open probability calculated assuming energetic additivity. The results suggest that the receptor is active in the presence of physiological concentrations of GABA and taurine, but, surprisingly, that receptor activity is only weakly potentiated by propofol.

Steady-state activation and modulation of the synaptic-type α1ß2γ2L GABAA receptor by combinations of physiological and clinical ligands.

The synaptic α1ß2γ2 GABAA receptor is activated phasically by presynaptically released GABA. The receptor is considered to be inactive between synaptic events when exposed to ambient GABA because of its low resting affinity to the transmitter. We tested the hypothesis that a combination of physiological and/or clinical positive allosteric modulators of the GABAA receptor with ambient GABA generates measurable steady-state activity. Recombinant α1ß2γ2L GABAA receptors were expressed in Xenopus oocytes and activated by combinations of low concentrations of orthosteric (GABA, taurine) and allosteric (the steroid allopregnanolone, the anesthetic propofol) agonists, in the absence and presence of the inhibitory steroid pregnenolone sulfate. Steady-state activity was analyzed using the three-state cyclic Resting-Active-Desensitized model. We estimate that the steady-state open probability of the synaptic α1ß2γ2L GABAA receptor in the presence of ambient GABA (1 µmol/L), taurine (10 µmol/L), and physiological levels of allopregnanolone (0.01 µmol/L) and pregnenolone sulfate (0.1 µmol/L) is 0.008. Coapplication of a clinical concentration of propofol (1 µmol/L) increases the steady-state open probability to 0.03. Comparison of total charge transfer for phasic and tonic activity indicates that steady-state activity can contribute strongly (~20 to >99%) to integrated activity from the synaptic GABAA receptor.