Flavan-3-ol esters: new agents for exploring modulatory sites on GABA(A) receptors

BACKGROUND AND PURPOSE

Enhancement of GABAergic function is the primary mechanism of important therapeutic agents such as benzodiazepines, barbiturates, neurosteroids, general anaesthetics and some anticonvulsants. Despite their chemical diversity, many studies have postulated that these agents may bind at a common or overlapping binding site, or share an activation domain. Similarly, we found that flavan-3-ol esters act as positive modulators of GABA_A receptors, and noted that this action resembled the in vitro profile of general anaesthetics. In this study we further investigated the interactions between these agents.

EXPERIMENTAL APPROACH

Using two-electrode voltage clamp electrophysiological recordings on receptors of known subunit composition expressed in Xenopus oocytes, we evaluated positive modulation by etomidate, loreclezole, diazepam, thiopentone, 5α-pregnan-3α-ol-20-one (THP) and the flavan-3-ol ester 2S,3R-trans 3-acetoxy-4′-methoxyflavan (Fa131) on wild-type and mutated GABA_A receptors.

KEY RESULTS

The newly identified flavan, 2S,3S-cis 3-acetoxy-3′,4′-dimethoxyflavan (Fa173), antagonized the potentiating actions of Fa131, etomidate and loreclezole at α1β2 and α1β2γ2L GABA_A receptors. Furthermore, Fa173 blocked the potentiation of GABA responses by high, but not low, concentrations of diazepam, but did not block the potentiation induced by propofol, the neurosteroid THP or the barbiturate thiopental. Mutational studies on ‘anaesthetic-influencing’ residues showed that, compared with wild-type GABA_A receptors, α1M236Wβ2γ2L and α1β2N265Sγ2L receptors are resistant to potentiation by etomidate, loreclezole and Fa131.

CONCLUSIONS AND IMPLICATIONS

Fa173 is a selective antagonist that can be used for allosteric modulation of GABA_A receptors. Flavan-3-ol derivatives are potential ligands for etomidate/loreclezole-related binding sites at GABA_A receptors and the low-affinity effects of diazepam are mediated via the same site.     

(+)- And (-)-borneol: efficacious positive modulators of GABA action at human recombinant alpha1beta2gamma2L GABA(A) receptors

(+)-Borneol is a bicyclic monoterpene used for analgesia and anaesthesia in traditional Chinese and Japanese medicine and is found in the essential oils of medicinal herbs, such as valerian. (+)-Borneol was found to have a highly efficacious positive modulating action at GABA(A) receptors, as did its enantiomer (-)-borneol. The effects of these bicyclic monoterpenes alone and with GABA were evaluated at recombinant human alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. (+)-Borneol (EC(50) 248microM) and (-)-borneol (EC(50) 237microM) enhanced the action of low concentrations of GABA by more than 1000%. These enhancing effects were highly dependent on the relative concentrations of the borneol enantiomer and GABA, and were insensitive to flumazenil indicating that (+)- and (-)-borneol were not acting at classical benzodiazepine sites. The maximal responses to GABA were enhanced 19% by (+)-borneol and reduced 21% by (-)-borneol. The borneol analogues isoborneol, (-)-bornyl acetate and camphor, produced less marked effects. At high concentrations (>1.5mM) (+)- and (-)-borneol directly activated GABA(A) receptors producing 89% and 84%, respectively, of the maximal GABA response indicative of a weak partial agonist action. Although of lower potency, the highly efficacious positive modulatory actions of (+)- and (-)-borneol on GABA responses were at least equivalent to that of the anaesthetic etomidate and much greater than that of diazepam or 5alpha-pregnan-3alpha-ol-20-one. The relatively rigid cage structure of these bicyclic monoterpenes and their high efficacy may aid in a greater understanding of molecular aspects of positive modulation of the activation of GABA(A) receptors.     

2'-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA(A) receptors

BACKGROUND AND PURPOSE

Flavonoids are known to have anxiolytic and sedative effects mediated via actions on ionotropic GABA receptors. We sought to investigate this further.

EXPERIMENTAL APPROACH

We evaluated the effects of 2′-methoxy-6-methylflavone (2′MeO6MF) on native GABA_A receptors in new-born rat hippocampal neurons and determined specificity from 18 human recombinant GABA_A receptor subtypes expressed in Xenopus oocytes. We used ligand binding, two-electrode voltage clamp and patch clamp studies together with behavioural studies.

KEY RESULTS

2′MeO6MF potentiated GABA at α2β1γ2L and all α1-containing GABA_A receptor subtypes. At α2β2/3γ2L GABA_A receptors, however, 2′MeO6MF directly activated the receptors without potentiating GABA. This activation was attenuated by bicuculline and gabazine but not flumazenil indicating a novel site. Mutation studies showed position 265 in the β1/2 subunit was key to whether 2′MeO6MF was an activator or a potentiator. In hippocampal neurons, 2′MeO6MF directly activated single-channel currents that showed the hallmarks of GABA_A Cl^- currents. In the continued presence of 2′MeO6MF the single-channel conductance increased and these high conductance channels were disrupted by the γ2(381–403) MA peptide, indicating that such currents are mediated by α2/γ2-containing GABA_A receptors. In mice, 2′MeO6MF (1–100 mg·kg⁻¹; i.p.) displayed anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark tests. 2′MeO6MF induced sedative effects at higher doses in the holeboard, actimeter and barbiturate-induced sleep time tests. No myorelaxant effects were observed in the horizontal wire test.

CONCLUSIONS AND IMPLICATIONS

2′MeO6MF will serve as a tool to study the complex nature of the activation and modulation of GABA_A receptor subtypes.     

GABA A receptors as in vivo substrate for the anxiolytic action of valerenic acid, a major constituent of valerian root extracts

Valerian extracts have been used for centuries to alleviate restlessness and anxiety albeit with unknown mechanism of action in vivo. We now describe a specific binding site on GABA_A receptors with nM affinity for valerenic acid and valerenol, common constituents of valerian. Both agents enhanced the response to GABA at multiple types of recombinant GABA_A receptors. A point mutation in the β2 or β3 subunit (N265M) of recombinant receptors strongly reduced the drug response. In vivo, valerenic acid and valerenol exerted anxiolytic activity with high potencies in the elevated plus maze and the light/dark choice test in wild type mice. In β3 (N265M) point-mutated mice the anxiolytic activity of valerenic acid was absent. Thus, neurons expressing β3 containing GABA_A receptors are a major cellular substrate for the anxiolytic action of valerian extracts.     

Allosteric interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) GABA(A) receptors

In a recent study we have provided evidence that inhibition of native GABA(A) receptors by zinc depends primarily on the allosteric modulation of receptor gating. Both the kinetics and the sensitivity of the GABA(A) receptor to zinc depend on subunit composition, especially on the presence of the gamma(2) subunit. To analyze the mechanism of action of zinc its effects have been tested on recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors expressed in HEK 293 cells. The currents produced by ultrafast application of GABA have been measured to assess the impact of zinc ions on GABA(A) receptor gating with resolution corresponding to the time scale of synaptic currents. While, as expected, zinc markedly reduced the peak amplitude of alpha(1)beta(2)-mediated currents, its effect on kinetics was significantly different from that observed for alpha(1)beta(2)gamma(2). In particular, unlike alpha(1)beta(2)gamma(2), zinc did not affect the onset of alpha(1)beta(2)-mediated responses. Moreover, zinc increased the extent of desensitisation of alpha(1)beta(2)gamma(2) receptors and reduced desensitisation of alpha(1)beta(2) ones. Quantitative analysis suggests that zinc exerts an allosteric modulation on both alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors. Zinc effects on alpha(1)beta(2)gamma(2) were qualitatively similar to those reported for native receptors.     

Homologous sites of GABA(A) receptor alpha(1), beta(3) and gamma(2) subunits are important for assembly

GABA(A) receptors are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta and one gamma subunit that assemble around an aqueous pore and form an intrinsic chloride ion channel. Using full-length or truncated chimeric subunits it was demonstrated that homologous sequences from different subunit classes, alpha(1)(54-68), beta(3)(52-66), and gamma(2)(67-81), are important for assembly of GABA(A) receptors composed of alpha(1), beta(3), and gamma(2) subunits. In addition, evidence was provided that these sequences all are located in topologically homologous regions of the different subunits. Finally, it was demonstrated that the sequences investigated cause a selective assembly with certain subunits only and thus influence subunit arrangement within GABA(A) 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.     

The novel anxiolytic ELB139 displays selectivity to recombinant GABA(A) receptors different from diazepam

A chemically heterogeneous group of compounds acts at the benzodiazepine (BZ) recognition site of the diverse gamma-aminobutyric acid type A (GABA(A)) receptor complexes which can assemble from more than 16 known subunits. Most 1,4-BZs like diazepam recognize all GABA(A)/BZ receptors containing the alpha1-3 or alpha5 together with any beta and the gamma2 subunit. Other compounds differentiate less, e.g. Ro15-4513, that additionally recognizes alpha4- and a6-containing receptors, or differentiate more, e.g. zolpidem, that recognizes preferentially alpha1-containing receptors. Here we describe the functional properties of 1-(4-chloro-phenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on (ELB139) in the presence and absence of the BZ receptor antagonist flumazenil (Ro15-1788) on recombinant alphaibeta2gamma2 (i=1-5) receptor subtypes expressed in HEK 293 cells. The properties were measured with the whole-cell variation of the patch-clamp technique and compared to those of diazepam. Like the latter, ELB139 did not potentiate GABA-induced currents in alpha4-containing receptors, but it displays functional subtype specificity between alpha1, alpha2, alpha3, and alpha5beta2gamma2 receptors with highest potency in alpha3-containing receptors but highest efficacy in alpha1- or alpha2-containing receptors, respectively. ELB139 acted as a partial agonist on these receptor subtypes reaching 40-50% of the efficacy of diazepam.     

3alpha,5beta-Reduced cortisol exhibits antagonist properties on cerebral cortical GABA(A) receptors

The tetrahydro-reduced derivatives of progesterone and deoxycorticosterone, allopregnanolone, and tetrahydrodeoxycorticosterone are potent positive modulators of GABA(A) receptors that are elevated by hypothalamic-pituitary-adrenal axis activation in rodents. In humans, 11-deoxycortisol and cortisol are important hypothalamic-pituitary-adrenal axis steroids. We hypothesized that C(3,5) reduction of 11-deoxycortisol and cortisol generates steroids with GABA(A) receptor activity. 3alpha,5beta-Reduced cortisol dose-dependently inhibited muscimol-stimulated chloride flux and tetrahydrodeoxycorticosterone potentiation of muscimol responses. Cortisol, 11-deoxycortisol, 5alpha-dihydrocortisol, 3alpha,5alpha-reduced cortisol, 3alpha,5alpha-reduced 11-deoxycortisol, and 3alpha,5beta-reduced 11-deoxycortisol had no activity at 1 muM and weaker negative modulatory activity at 10 muM. We conclude that cortisol metabolism may produce antagonistic GABAergic activity.     

Altered response to benzodiazepine anxiolytics in mice lacking GABA B(1) receptors

Recently, we demonstrated that mice lacking the GABA(B(1)) subunit were more anxious than wild-type animals in several behavioural paradigms, most notably in the light-dark test. In an attempt to assess the effects of classical benzodiazepine anxiolytics on anxiety-like behaviour observed in these mice, animals were administered either chlordiazepoxide (10 mg/kg, p.o.) or diazepam (7.5 mg/kg, p.o.) prior to testing in the light-dark box. Surprisingly, in contrast with the wild-type mice, neither benzodiazepines decreased anxiety-like behaviour in GABA(B(1))(-/-) mice. These data suggest that targeted deletion of GABA(B(1)) subunit alters GABA(A) receptor function in vivo.     

delta-Subunit containing GABAA receptor knockout mice are less sensitive to the actions of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol

The pharmacological profile of a gamma-aminobutyric acid A (GABA(A)) receptor depends upon subunit composition. Studies using recombinant expression systems suggest that delta-subunit containing GABA(A) receptors are particularly sensitive to the actions of the GABA(A) partial agonist, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP, gaboxadol). Here we investigated the actions of THIP in mutant mice lacking the GABA(A) receptor delta-subunit gene. Using the chloride flux assay, we determined that the actions of THIP were reduced by 21% in the cortical, but not cerebellar, membranes of knockout mice. Similar results were seen with another GABA(A) agonist, muscimol. Moreover, delta-subunit knockout mice exhibited a 54% reduction in sensitivity to the hypnotic actions of THIP as assessed by the loss of righting reflex test. These data support the notion that delta-containing GABA(A) receptors are at least partially responsible for the actions of THIP, and contribute to the growing literature suggesting that the pharmacological specificity of GABA(A) receptors depends on which subunits are present or absent.     

Chronic blockade of cannabinoid CB2 receptors induces anxiolytic-like actions associated with alterations in GABA(A) receptors

BACKGROUND AND PURPOSE

The aim of this study was to explore the effects of CB₂ receptor agonist and antagonist in the regulation of anxiety-like behaviours.

EXPERIMENTAL APPROACHES

Effects of acute and chronic treatment with the CB₂ receptor agonist JWH133 and CB₂ receptor antagonist AM630 were evaluated in the light-dark box (LDB) and elevated plus maze (EPM) tests in Swiss ICR mice. CB₂ receptor, GABA_Aα₂ and GABA_Aγ₂ gene and protein expression in the cortex and amygdala of mice chronically treated with JWH133 or AM630 were examined by RT-PCR and Western blot. Effects of chronic AM630 treatment were evaluated in spontaneously anxious DBA/2 mice in LDB.

KEY RESULTS

Acute JWH133 treatment failed to produce any effect. Acute AM630 treatment increased anxiety and was blocked by pre-treatment with JWH133. Chronic JWH133 treatment increased anxiety-like behaviour whereas chronic AM630 treatment was anxiolytic in LDB and EPM tests. Chronic AM630 treatment increased gene and reduced protein expression of CB₂ receptors, GABA_Aα₂ and GABA_Aγ₂ in cortex and amygdala. Chronic JWH133 treatment resulted in opposite gene and protein alterations. In addition, chronic AM630 administration decreased the anxiety of DBA/2 mice in the LDB test.

CONCLUSIONS AND IMPLICATIONS

The opposing behavioural and molecular changes observed after chronic treatment with AM630 or JWH133 support the key role of CB₂ receptors in the regulation of anxiety. Indeed, the efficacy of AM630 in reducing the anxiety of the spontaneously anxious DBA/2 strain of mice strengthens the potential of the CB₂ receptor as a new target in the treatment of anxiety-related disorders.     

The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat alpha1beta2gamma2L GABA(A) receptor

Background and purpose:

Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABA_A receptors in the brain. In this study, we have examined the modulation of the common brain GABA_A receptor subtype by fipronil and its major metabolite, fipronil sulphone.

Experimental approach:

Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat α1β2γ2L GABA_A receptors.

Key results:

The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The α1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the α1β2γ2L receptor.

Conclusions and implications:

We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain.     

Isoflurane-induced surgical tolerance mediated only in part by beta3-containing GABA(A) receptors

The targets which mediate the actions of the volatile general anaesthetic isoflurane are unknown. Based on pharmacological studies using GABA(A) receptor antagonists it has recently been suggested that GABA(A) receptors would not mediate the immobilizing action of isoflurane. Using the beta3(N265M) knock-in mouse model we found that the mutant mice were less sensitive to the immobilizing action of isoflurane, indicating a role of beta3-containing GABA(A) receptors in mediating immobility. At high concentrations isoflurane also immobilizes beta3(N265M) mice, indicating that other targets also mediate immobility. Thus, our findings support a multisite model for the immobilizing action of isoflurane.     

The influence of subunit composition on the interaction of neurosteroids with GABA(A) receptors

The influence of the subunit composition of human GABA(A) receptors upon the GABA-modulatory properties of 5alpha-pregnan-3alpha-ol-20-one (5alpha,3alpha) has been examined using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. Steroid potency (EC(50)) is modestly influenced by the alpha-isoform (alpha(x)beta(1)gamma(2L); x=1-6). alpha(2)-, alpha(4)- and alpha(5)-containing receptors are significantly less sensitive to the action of low concentrations of 5alpha,3alpha (10-100 nM) when compared to alpha(1,3,6)beta(1)gamma(2L) receptors. Additionally, the maximal effect of the steroid is favoured at alpha(6)-containing receptors. The beta-isoform (alpha(1)beta(y)gamma(2L); y=1-3) has little influence on the GABA-modulatory effect of the neurosteroid. The EC(50) of 5alpha,3alpha is only modestly influenced by the omission of the gamma(2) subunit (alpha(1)beta(1)gamma(2L) vs alpha(1)beta(1)): while the maximal effect is favoured by the binary complex. However, the identity of the gamma subunit influences the GABA(A)-modulatory potency of 5alpha,3alpha with gamma(2)- and gamma(1)-containing receptors being the most and the least sensitive to 5alpha,3alpha, respectively. Finally, incorporation of the epsilon, or delta subunit dramatically reduces and augments the GABA-enhancing actions of the steroid, respectively. These findings provide evidence that 5alpha,3alpha discriminates amongst recombinant receptors of varied subunit composition. Furthermore, this selectivity may contribute to their neuronal specificity and behavioural profile.     

Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABA(A) receptors in vitro

BACKGROUND AND PURPOSE

Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA_A receptors (GABA_ARs). We previously reported that trans-6,4′-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA_ARs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4′-methoxyretrochalcone (Rc-Br) and 4,3′-dimethoxychalcone (Ch-OMe) on GABA_ARs.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp recordings were made to determine the effects of these derivatives on GABA_ARs expressed in HEK-293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain.

KEY RESULTS

Rc-OMe strongly potentiated GABA-evoked currents at recombinant α_1–4β₂γ_2s and α₄β₃δ receptors but much less at α₁β₂ and α₄β₃. Rc-Br and Ch-OMe potentiated GABA-evoked currents at α₁β₂γ_2s. The potentiation by Rc-OMe was only reduced at α₁H101Rβ₂γ_2s and α₁β₂N265Sγ_2s, mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc-OMe and pentobarbital as well as by Rc-OMe and the neurosteroid 3α,21-dihydroxy-5α-pregnan-20-one was supra-additive. Rc-OMe modulation exhibited no apparent voltage-dependence, but was markedly dependent on GABA concentration. In neurones, Rc-Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc-OMe and Rc-Br positively modulated synaptic and extrasynaptic diazepam-insensitive GABA_ARs.

CONCLUSIONS AND IMPLICATIONS

The trans-retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA_ARs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA_A-related disorders in vivo.     

In vivo labelling of alpha5 subunit-containing GABA(A) receptors using the selective radioligand [(3)H]L-655,708

L-655,708 is an imidazobenzodiazepine possessing 30-70-fold selectivity for the benzodiazepine binding site of GABA_A receptors containing an α5 rather than α1, α2 or α3 subunit. In the present study, [³H]L-655,708 was used to label mouse brain benzodiazepine binding sites in vivo. When compared to inhibition of in vivo binding of the non-selective ligand [³H]Ro 15-1788, the pharmacology of mouse in vivo [³H]L-655,708 binding was consistent with selective in vivo labelling of α5 subunit-containing GABA_A receptors. Thus, diazepam was equipotent at inhibiting in vivo [³H]L-655,708 and [³H]Ro 15-1788 binding; zolpidem, which has very low affinity for α5-containing GABA_A receptors, gave no inhibition of in vivo [³H]L-655,708 binding despite inhibiting in vivo [³H]Ro 15-1788 binding; and L-655,708 was more potent at inhibiting the in vivo binding of [³H]L-655,708 compared to [³H]Ro 15-1788. This pharmacological specificity of in vivo [³H]L-655,708 binding was confirmed autoradiographically. Hence, the anatomical distribution of in vivo [³H]L-655,708 binding was comparable to the distribution of α5-containing GABA_A receptors identified in vitro. Moreover, this distribution was distinct from that identified using [³H]Ro 15-1788. These data therefore suggest that [³H]L-655,708 can be used to identify α5-containing GABA_A receptors in vivo and that this ligand can be used to measure receptor occupancy of α5-selective ligands.     

Role of the alpha subunit in the modulation of GABA(A) receptors by anabolic androgenic steroids

Neural transmission mediated by circuits expressing α₂ subunit-containing γ-aminobutyric acid type A (GABA_A) receptors is critical for the expression of behaviors known to be altered by anabolic androgenic steroids (AAS). Here we show that micromolar concentrations of AAS, which reflect levels found in steroid abusers, induce positive modulation of currents from α₂β₃γ_2L recombinant receptors elicited by pulses of GABA that mimic synaptic conditions in a manner that is mechanistically distinct from modulation induced at α₁β₃γ_2L receptors. Specifically, at α₂-containing receptors, the AAS, 17α-methyltestosterone (17α-MeT) enhanced peak current, slowed deactivation, diminished desensitization, and promoted entry of receptors into more distal states along the activation pathway. Analysis of GABA_A receptor-mediated synaptic currents in primary cortical neurons followed by single cell real-time RT-PCR demonstrated that 17α-MeT enhancement of synaptic currents is proportional to the ratio of α₂ to α₁ subunit mRNA. Finally, we show that the modulation elicited by AAS is not comparable to that produced by micromolar concentrations of other positive allosteric modulators at α₂-containing receptors. In sum, these data indicate that AAS elicit effects on GABA_A receptor function that depend significantly on α subunit composition and that the mechanism of AAS modulation of GABA_A receptors is distinct from that of other positive allosteric modulators.     

A single point mutation of the GABA(A) receptor alpha5-subunit confers fluoxetine sensitivity

Fluoxetine has been reported to be a novel allosteric modulator of GABA(A) receptors with the notable exception of receptors that contain the alpha5-subunit isoform [Robinson, R.T., Drafts, B.C., Fisher, J.L., 2003. Fluoxetine increases GABA(A) receptor activity through a novel modulatory site. J. Pharmacol. Exp. Ther. 304, 978-984]. A mutagenic strategy has been used to investigate the structural basis for the insensitivity of this subunit. An alpha1/alpha5-subunit chimeragenesis approach first demonstrated the importance of the alpha1-subunit N-terminal sequence E165-D183 (corresponding to alpha5 E169-D187) in fluoxetine modulation. Specific amino acid substitutions in this domain subsequently revealed that a single mutation in the alpha5-subunit to the equivalent residue in alpha1 (T179A) was sufficient to confer fluoxetine sensitivity to the alpha5-containing receptor. However, the reciprocal mutation in the alpha1-subunit (A175T) did not result in a loss in sensitivity, suggesting the involvement of additional determinants for fluoxetine modulation. A comparative modeling approach was used to probe amino acids that may lie in close proximity to alpha1A175. This led serendipitously to the identification of a specific residue, alpha1F45, which, when mutated to an alanine, resulted in a significant decrease in potency for activation of the receptor by GABA and also reduced the efficacies of the partial agonists, THIP and P4S.     

Biochemical identification of the binding domain in the GABA(A) receptor-associated protein (GABARAP) mediating dimer formation

The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and clustering of GABA(A) receptors. GABARAP interacts with microtubules and the gamma2 subunit of GABA(A) receptor and modulates channel kinetics. From crystal structures of GABARAP in high salt concentration it has been proposed that oligomerization of GABARAP might take place in a head-to-tail fashion. In this study, we report that GABARAP self-associates and dimerizes in physiological salt concentrations. We find no evidence for higher order complex larger than a dimer. By using deletion constructs of GABARAP we show that interaction takes place between amino acid 36 and 68. We further narrow the interacting domain by inhibiting the self-association, by adding GABARAP-derived synthetic peptides in GST pull-down assays and shows that the interaction specifically takes place in the previously identified GABARAP-GABA(A) receptor interaction domain from amino acid 41-51. The identification of binding domains in GABARAP allows for the study of GABARAP functions, including GABA(A) receptor dynamics.