Conserved site for neurosteroid modulation of GABA A receptors

This study addresses whether the potentiation site for neurosteroids on GABA(A) receptors is conserved amongst different GABA(A) receptor isoforms. The neurosteroid potentiation site was previously identified in the alpha1beta2gamma2S receptor by mutation of Q241 to methionine or leucine, which reduced the potentiation of GABA currents by the naturally occurring neurosteroids, allopregnanolone or tetrahydrodeoxycorticosterone (THDOC). By using heterologous expression of GABA(A) receptors in HEK cells, in combination with whole-cell patch clamp recording methods, a relatively consistent potentiation by allopregnanolone of GABA-activated currents was evident for receptors composed of one alpha subunit isoform (alpha2-5) assembled with beta3 and gamma2S subunits. Using mutant alphabetagamma receptors, the neurosteroid potentiation was universally dependent on the conserved glutamine residue in M1 of the respective alpha subunit. Studying wild-type and mutant receptors composed of alpha4beta3delta subunits revealed that the delta subunit is unlikely to contribute to the neurosteroid potentiation binding site and probably affects the efficacy of potentiation. Thus, in keeping with the ability of neurosteroids to potentiate GABA currents via a broad variety of GABA(A) receptor isoforms in neurons, the potentiation site is structurally highly conserved on this important neurotransmitter receptor family.     

Auto-modulation of neuroactive steroids on GABA A receptors: a novel pharmacological effect

GABA(A) receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA(A) receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (alpha1beta3delta, alpha1beta3gamma2L, alpha6beta3delta, alpha6beta3gamma2L) and at GABA(A) receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5alpha-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta subtypes while it enhanced potentiation at alpha1beta3delta isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA(A) receptor sites containing the delta subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.     

Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant alpha1beta2gamma2 GABAA receptors

Benz[e]indenes are tricyclic analogs of neuroactive steroids and can be modulators of GABA(A) receptor activity. We have examined the mechanisms of action of the benz[e]indene compound [3S-(3alpha,3aalpha,5abeta,7beta,9aalpha,9bbeta)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch-clamp and whole-cell recordings from human embryonic kidney cells transfected with rat GABA(A) receptor alpha1, beta2, and gamma2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA(A) receptor activity with a peak effect at 2 microM. The mechanism of modulation is similar but not identical to that of neuroactive steroids. Similar to steroids, BI-2 acts by prolonging the mean open time duration through an effect on the duration and prevalence of the longest open time component. However, in contrast to many steroids, BI-2 does not selectively reduce the channel closing rate. The potentiating action of BI-2 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the alpha1 subunit Q242W and the double mutation alpha1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 microM), BI-2 inhibits receptor function by enhancing the apparent rate of desensitization. From single-channel recordings, we estimate that the entry rate into the inhibited or blocked state, k(+B), is 0.50 microM(-1) s(-1). Based on the kinetic mechanism of action, and the finding that this effect is blocked by the alpha1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.     

Multiple structural features of steroids mediate subtype-selective effects on human alpha4beta3delta GABAA receptors

Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory gamma-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the delta subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional delta-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk-) cells stably transfected with human GABAA receptor alpha4beta3delta and alpha4beta3gamma2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the alpha4beta3delta receptor. 5beta-Pregnan-3beta-ol-20-one, 5beta-pregnane-3alpha,20beta-diol and 5beta-pregnane-3alpha,17alpha-diol-11,20-dione showed particularly high efficacy for alpha4beta3delta. No compounds were identified that simply inhibited responses at delta-containing receptors. However, 5beta-pregnane-3alpha,17alpha,20beta-triol, prednisolone 21-acetate, 4-pregnene-17alpha,20alpha-diol-3-one-20-acetate, 4-pregnen-20alpha-ol-3-one, and 5beta-pregnane-3alpha,17alpha,21-triol-20-one inhibited, though did not abolish, GABA responses at the alpha4beta3gamma2 subtype, while evoking modest-amplitude potentiation of alpha4beta3delta responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of delta-containing GABAA receptors.     

Mechanisms of anabolic androgenic steroid modulation of alpha(1)beta(3)gamma(2L) GABA(A) receptors

Modulation of GABA(A) receptors induced by both anabolic androgenic steroids (AAS) and the benzodiazepine (BZ) site agonist, zolpidem, show equivalent dependence upon gamma subunit composition suggesting that both compounds may be acting at a shared allosteric site. Here we have characterized modulation induced by the AAS, 17alpha-methyltestosterone (17alpha-MeT), for responses elicited from alpha(1)beta(3)gamma(2L) GABA(A) receptors and compared it to modulation induced by the BZ site agonists, zolpidem and diazepam. For responses elicited by brief pulses of 20 microM GABA, both the AAS and the BZ site compounds significantly increased the peak current amplitudes and total charge transfer, although 17alpha-MeT was an appreciably weaker agonist than either diazepam or zolpidem at alpha(1)beta(3)gamma(2L) receptors. Neither class of modulator enhanced peak current amplitudes for responses elicited by mM concentrations of GABA. BZ site compounds altered time constants of deactivation, desensitization, and recovery from desensitization, however 17alpha-MeT had no overall effect on these parameters. Experiments in which 17alpha-MeT and BZ site ligands were applied concomitantly indicated that potentiation elicited by 17alpha-MeT and zolpidem were additive and that potentiation by 17alpha-MeT could be elicited in the presence of concentrations of flumazenil that blocked BZ potentiation. Finally, kinetic modeling suggests that while effects of 17alpha-MeT can be simulated by altering receptor affinity, the data for these alpha(1)beta(3)gamma(2L) receptors were best fitted by simulations in which 17alpha-MeT increases transitions into the singly liganded open state. Taken together, our results suggest that 17alpha-MeT does not act at the high-affinity BZ site, but may elicit some of its effects at the low affinity BZ site or at a novel site.     

Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor with the intravenous agent, propofol

Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol > cyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor.     

Redox modulation of GABAA receptors obscured by Zn2+ complexation

Redox reagents are thought to modulate gamma-Aminobutyric acid type A (GABA(A)) receptors by regulating the redox state of the N-terminal disulphide bridge. Examining the redox sensitivity of recombinant GABA(A) receptors in human embryonic kidney cells, using whole-cell patch clamp techniques, revealed that alpha1beta2(H267A) and alpha1beta2gamma2 receptors, which are both less sensitive to Zn(2+) and H(+) modulation, ablated the potentiating effect of the reducing agent, dithiothreitol (DTT) seen for alpha1beta2 receptors. This effect could result from disruption to the redox signal transduction pathway or be due to DTT chelating Zn(2+) from its H267 inhibitory binding site, consequently potentiating GABA-activated currents in alpha1beta2 but not alpha1beta2(H267A) or alpha1beta2gamma2 receptors. A Zn(2+) chelating agent, tricine, potentiated GABA currents for the alphabeta constructs and vertically displaced GABA dose-response curves, suggesting that these receptors are subject to some inhibition by basal Zn(2+). Tricine, did not affect the GABA currents of either alpha1beta2(H267A) or alpha1beta2gamma2 receptors but did prevent the potentiation by 2 mM DTT and reduced the potentiation caused by 10 mM DTT on alpha1beta2 receptors. Thus, at low concentrations of DTT, a substantial component of the potentiation probably occurs via Zn(2+) chelation from H267 in the ion channel. In contrast, at higher DTT concentrations, it is more likely to be acting as a redox agent, which modulates both alphabeta and alphabetagamma subunit receptors.     

The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores

Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.     

Flavan-3-ol derivatives are positive modulators of GABA(A) receptors with higher efficacy for the alpha(2) subtype and anxiolytic action in mice

Recent genetic and pharmacological studies have demonstrated that alpha(2)-containing GABA(A) receptors mediate the anxiolytic effects of benzodiazepines, setting a new strategy in developing novel, non-sedative anxiolytic agents. In this study we show that stereoisomers of 3-acetoxy-4'-methoxyflavan are positive modulators of recombinant alpha(1,2,3,5)beta(2)gamma(2L) and alpha(1)beta(2) GABA(A) receptors expressed in Xenopus laevis oocytes. GABA(C) receptors are insensitive to modulation by these compounds. In each case, the enhancement was evident at low micromolar concentrations and occurred independently of the classical high affinity benzodiazepine site, as it could not be blocked by the antagonist flumazenil. Importantly, the compound Fa131 was significantly more efficacious at enhancing GABA-induced currents (EC(5)) at alpha(2)beta(2)gamma(2L) receptors compared to alpha(1)beta(2)gamma(2L), alpha(3)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors (E(max)=21.0+/-1.7 times, compared to 8.5+/-0.7 times at alpha(1)-, 9.5+/-0.6 times at alpha(3)- and 5.2+/-0.4 times at alpha(5)-contaning GABA(A) receptors), suggesting a potential use as an anxiolytic. In mice, this agent (1-30mg/kg i.p.) induced anxiolytic-like action in two unconditioned models of anxiety: the elevated plus maze and the light/dark paradigms. No sedative or myorelaxant effects were detected using the hole board, actimeter and horizontal wire tests, and only weak barbiturate-potentiating effects on the loss of righting reflex test. Fa131 demonstrated improved segregation of anxiolytic and sedative doses when compared to the non-selective agonist diazepam. Finally, flavan derivatives highlight the potential of targeting non-benzodiazepine allosteric sites in the search for new anxioselective drugs.     

Structurally diverse amphiphiles exhibit biphasic modulation of GABAA receptors: similarities and differences with neurosteroid actions

Background and purpose:

Some neurosteroids, notably 3α-hydroxysteroids, positively modulate GABA_A receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions.

Experimental approach:

Xenopus laevis oocytes were challenged with Triton X-100, octyl-β-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations.

Key results:

These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 µM) promoted potentiation and a high concentration (20 µM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (α1V256S) and positive (α1Q241L or α1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in α1V256S-mutated receptors, but potentiation was unaffected. In α1Q241L- or α1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact.

Conclusions and implications:

Structurally diverse amphiphiles have antagonist actions at GABA_A receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroid-positive modulators. Thus, such antagonism at GABA_A receptors does not have a clear pharmacophore requirement.     

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors

1: The pharmacology of the stable cell line expressing human alpha(4)beta(3)delta GABA(A) receptor was investigated using whole-cell patch-clamp techniques. 2: alpha(4)beta(3)delta receptors exhibited increased sensitivity to GABA when compared to alpha(4)beta(3)gamma(2) receptors, with EC(50)'s of 0.50 (0.46, 0.53) microM and 2.6 (2.5, 2.6) microM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at alpha(4)beta(3)delta receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. 3: The delta subunit conferred slow desensitization to GABA, with rate constants of 4.8+/-0.5 s for alpha(4)beta(3)delta and 2.5+/-0.2 s for alpha(4)beta(3)gamma(2). However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. 4: alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2) demonstrated equal sensitivity to inhibition by the cation zinc (2-3 microM IC(50)). However, alpha(4)beta(3)delta receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC(50) for GABA antagonists SR-95531 and picrotoxin, was similar for alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2). Likewise, inhibition was observed on both subtypes at high and low pH. 5: alpha(4)beta(3)delta receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on alpha(4)beta(3)gamma(2) cells, and the inverse agonist DMCM showed allosteric inhibition of alpha(4)beta(3)gamma(2) receptors. 6: The efficacy of neurosteroids at alpha(4)beta(3)delta receptors was greatly enhanced over that observed at alpha(4)beta(3)gamma(2) receptors. The greatest effect was observed using THDOC with 524+/-71.6% potentiation at alpha(4)beta(3)delta and 297.9+/-49.7% at alpha(4)beta(3)gamma(2) receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at alpha(4)beta(3)delta receptors versus alpha(4)beta(3)gamma(2) receptors. 7: We show that the alpha(4)beta(3)delta receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965-974     

Selective antagonism of 5alpha-reduced neurosteroid effects at GABA(A) receptors

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.     

The relative amount of cRNA coding for gamma2 subunits affects stimulation by benzodiazepines in GABA(A) receptors expressed in Xenopus oocytes

Benzodiazepine (BZD) potentiation of GABA-activated Cl(-)-current (I(GABA)) in recombinant GABA(A) receptors requires the presence of the gamma subunit. When alpha1, beta2 and gamma2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of I(GABA) is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the gamma2S subunit. In addition, GABA EC(50) values for alpha1beta2gamma2 (1:1:1) receptors are intermediate to values measured for alpha1beta2 (1:1) and alpha1beta2gamma2 (1:1:10) receptors. We conclude that co-expression of equal ratios of alpha1, beta2 and gamma2 subunits in Xenopus oocytes produces a mixed population of alpha1beta2 and alpha1beta2gamma2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of gamma2 subunit cRNA relative to alpha1 and beta2 cRNA. This results in a purer population of alpha1beta2gamma2 receptors.     

Effects of gamma-HCH and delta-HCH on human recombinant GABA(A) receptors: dependence on GABA(A) receptor subunit combination

1. Human GABA(A) receptors containing different alpha and beta subunits with or without the gamma 2S or gamma 2L subunits were expressed in XENOPUS: oocytes and the effects of the insecticides gamma- and delta-hexachlorocyclohexane (gamma-HCH and delta-HCH, respectively) on these receptor subunit combinations were examined using two electrode voltage-clamp procedures. 2. gamma-HCH produced incomplete inhibition of GABA responses on all receptor combinations examined with affinities in the range of 1.1--1.9 microM. Affinity was not dependent on subunit composition but the maximum percentage of inhibition was significantly reduced in beta 1-containing receptors. delta-HCH both potentiated GABA(A) receptors and activated them in the absence of GABA at concentrations higher than those producing potentiation. Allosteric enhancement of GABA(A) receptor function by delta-HCH was not affected by the subunit composition of the receptor, By contrast the GABA mimetic actions of delta-HCH were abolished in receptors containing either alpha 4, beta 1 or gamma 2L subunits. 4. Sensitivity to the direct actions were not restored in receptors containing the mutant beta 1(S290N) subunit, but alpha 1 beta 2 gamma 2L receptors became sensitive to the direct actions of delta-HCH when oocytes were treated for 24 h with the protein kinase inhibitor isoquinolinesulphonyl-2-methyl piperazine dihydrochloride (H-7). 5. We have shown the influence of various alpha, beta and gamma subunits on the inhibitory, GABA mimetic and allosteric effects of HCH isomers. The data reveal that neither the inhibitory actions of gamma-HCH nor the allosteric effects delta-HCH has a strict subunit dependency. By contrast, sensitivity to the direct actions of delta-HCH are abolished in receptors containing alpha 4, beta 1 or gamma 2L subunits.     

High affinity, heterogeneous displacement of [3H]EBOB binding to cerebellar GABA A receptors by neurosteroids and GABA agonists

Heterogeneous binding interactions of cerebellar GABA(A) receptors were investigated with GABA agonists and neurosteroids. GABA(A) receptors of rat cerebellum were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB), a convulsant radioligand. Saturation analysis revealed a homogenous, nanomolar population of [(3)H]EBOB binding. Both GABA and 5alpha-tetrahydrodeoxycorticosterone (5alpha-THDOC) displaced [(3)H]EBOB binding heterogeneously, with nanomolar and micromolar potencies. The nanomolar phase of displacement by GABA was selectively abolished by 100 microM furosemide. Physiological concentrations of allopregnanolone (8 nM) and 5alpha-THDOC (20 nM) increased the displacing effects of nanomolar GABA. GABA (0.3 microM ) and 5alpha-THDOC (0.3 microM ) potentiated the micromolar population of displacement by the other. Taurine inhibited [(3)H]EBOB binding also heterogeneously, with micromolar and millimolar potencies, and 0.3 microM 5alpha-THDOC potentiated this inhibition. 5beta-THDOC did not affect [(3)H]EBOB binding significantly but in 1 microM it antagonised selectively the nanomolar displacement by 5alpha-THDOC. [(3)H]EBOB binding to hippocampal GABA(A) receptors was inhibited by GABA and allopregnanolone with low (micromolar) potencies and with slope values higher than unity referring to allosteric interaction. High affinity displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be associated with constitutively open alpha(6)betadelta GABA(A) receptors, tonic GABAergic inhibitory neurotransmission and its modulation by physiological concentrations of neurosteroids.     

Receptor subtype-dependent positive and negative modulation of GABA(A) receptor function by niflumic acid,a nonsteroidal anti-inflammatory drug

In addition to blocking cyclooxygenases, members of the fenamate group of nonsteroidal anti-inflammatory drugs have been proposed to affect brain GABAA receptors. Using quantitative autoradiography with GABAA receptor-associated ionophore ligand [35S]t-butylbicyclophosphorothionate (TBPS) on rat brain sections, one of the fenamates, niflumate, at micromolar concentration was found to potentiate GABA actions in most brain areas, whereas being in the cerebellar granule cell layer an efficient antagonist similar to furosemide. With recombinant GABAA receptors expressed in Xenopus laevis oocytes, we found that niflumate potentiated 3 microM GABA responses up to 160% and shifted the GABA concentration-response curve to the left in alpha1beta2gamma2 receptors, the predominant GABAA receptor subtype in the brain. This effect needed the gamma2 subunit, because on alpha1beta2 receptors, niflumate exhibited solely an antagonistic effect at high concentrations. The potentiation was not abolished by the specific benzodiazepine site antagonist flumazenil. Niflumate acted as a potent antagonist of alpha6beta2 receptors (with or without gamma2 subunit) and of alphaXbeta2gamma2 receptors containing a chimeric alpha1 to alpha6 subunit, which suggests that niflumate antagonism is dependent on the same transmembrane domain 1- and 2-including fragment of the alpha6 subunit as furosemide antagonism. This antagonism was noncompetitive because the maximal GABA response, but not the potency, was reduced by niflumate. These data show receptor subtype-dependent positive and negative modulatory actions of niflumate on GABAA receptors at clinically relevant concentrations, and they suggest the existence of a novel positive modulatory site on alpha1beta2gamma2 receptors that is dependent on the gamma2 subunit but not associated with the benzodiazepine binding site.     

Natural and enantiomeric etiocholanolone interact with distinct sites on the rat alpha1beta2gamma2L GABAA receptor

We have studied the ability of the androgen etiocholanolone and its enantiomer (ent-etiocholanolone) to modulate rat alpha1beta2gamma2L GABA(A) receptor function transiently expressed in human embryonic kidney cells. Studies on steroid enantiomer pairs can yield powerful new information on the pharmacology of steroid interactions with the GABA(A) receptor. Both steroids enhance currents elicited by GABA, but ent-etiocholanolone is much more powerful than etiocholanolone at producing potentiation. At a low GABA concentration (0.5 microM, 相似文献   

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions

BACKGROUND AND PURPOSE: Neuroactive steroids are potent modulators of GABA(A) receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABA(A) receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABA(A) receptor, such as (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP, allopregnanolone). EXPERIMENTAL APPROACH: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between gamma-cyclodextrin and neuroactive steroids of different structural classes. KEY RESULTS: Both a bioassay based on electrophysiological assessment of GABA(A) receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that gamma-cyclodextrin sequesters steroids rather than directly influencing GABA(A) receptor function. Neither a 5beta-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and gamma-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and gamma-cyclodexrin ranged from 10-60 microM. Although gamma-cyclodextrin accommodates a range of natural and synthetic steroids, C(11) substitutions reduced inclusion complex formation. Using gamma-cyclodextrin to remove steroid not directly bound to GABA(A) receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3alpha- hydroxysteroids but not inhibition by sulphated steroids. CONCLUSIONS AND IMPLICATIONS: We conclude that gamma-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids.     

alpha1beta2delta, a silent GABAA receptor: recruitment by tracazolate and neurosteroids

BACKGROUND AND PURPOSE: This study investigated the alpha(1)beta(2)delta isoform of the GABA(A) receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown. EXPERIMENTAL APPROACH: We expressed alpha(1)beta(2)delta GABA(A) receptors in Xenopus laevis oocytes. GABA-activated currents, in the presence and absence of modulators, were recorded using the two-electrode voltage clamp technique. KEY RESULTS: The alpha(1)beta(2)delta isoform of the GABA(A) receptor exhibited an extremely small GABA-mediated current. Tracazolate increased the current amplitude evoked by a half-maximal concentration (EC(50)) of GABA by 59-fold. The maximum current was increased 23-fold in the presence of a saturating GABA concentration. Concomitant with the increase in the maximum, was a 4-fold decrease in the EC(50). Finally, a mutation in the second transmembrane domain of the delta subunit that increases receptor efficacy (L286S), eliminated the increase in the maximum GABA-activated current. The endogenous neurosteroid, tetrahydrodeoxycorticosterone (THDOC), also decreased the EC(50) and increased the maximum current amplitude, although to a lesser degree than that of tracazolate. CONCLUSIONS AND IMPLICATIONS: Taken all together, these findings indicate that the small GABA-mediated currents in the absence of the modulator are due to a low efficacy for activation. In the absence of modulators, alpha(1)beta(2)delta GABA receptors would be effectively silent and therefore contribute little to inhibition in the CNS. In the presence of tracazolate or endogenous neurosteroids however, this particular receptor isoform could exert a profound inhibitory influence on neuronal activity.     

Molecular mechanisms of interaction between the neuroprotective substance riluzole and GABAA-receptors

The antiepileptic drug riluzole is used as a therapeutic agent in amyotrophic lateral sclerosis due to its neuroprotective effects. Besides presynaptic inhibition of GABAergic and preferentially glutamatergic transmission, it also potentiates postsynaptic GABA(A)-receptor function. We investigated the postsynaptic effects of riluzole on GABA(A)-receptor channels by use of the patch-clamp technique. Recombinant alpha(1)beta(2)gamma(2s) and alpha(1)beta(2) GABA(A) receptors were expressed in HEK 293 cells by transient transfection. Pulses of GABA were applied in combination with different concentrations of riluzole to whole cell or outside-out patches with either alpha(1)beta(2)gamma(2s) or alpha(1)beta(2) GABA(A)-receptor channels. Co-application of riluzole led to a slight decrease of absolute peak current amplitudes and steady-state currents in prolonged presence of GABA at saturating concentrations. In the presence of riluzole, enhancement of current amplitudes was observed with lower concentrations of GABA at alpha(1)beta(2)gamma(2s) receptors and to a lower extent also at alpha(1)beta(2) receptors. Thus, the potentiating effect of riluzole was shown to be not abolished in the absence of the gamma2s-subunit. A further prominent effect of riluzole was a highly significant acceleration of the time course of current decay, most probably pointing to an open-channel block-like mechanism of action. As both receptor subtypes were affected similarly by the block, it could be concluded that the respective binding sites should be assumed within a region of high sequence homology like it is given for the channel-lining M2 domain of GABA(A)-receptor subunits. In conclusion, three different molecular mechanisms of interaction of the neuroprotective compound riluzole were observed at two different subtypes of GABA(A) receptor channels. The results further point to the impact of the inhibitory as well as the excitatory synaptic activity as a pharmacological target to counteract chronic excitotoxicity and reveal molecular mechanisms of action of the only one neuroprotective drug in current clinical use in patients suffering from amyotrophic lateral sclerosis.