Delia Belelli

The influence of subunit composition on the interaction of neurosteroids with GABAA 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.

Extrasynaptic GABAA Receptors of Thalamocortical Neurons: A Molecular Target for Hypnotics

Among hypnotic agents that enhance GABAA receptor function, etomidate is unusual because it is selective for β2/β3 compared with β1 subunit-containing GABAA receptors. Mice incorporating an etomidate-insensitive β2 subunit (β2N265S) revealed that β2 subunit-containing receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the β2-containing receptors that mediate these actions is unknown, the thalamus is implicated. We have characterized GABAA receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, β–/–2, and β2N265S mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes ∼80% of the total GABAA receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the β2N265S mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nm to 3 μm) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

The GABA modulating and GABA‐mimetic actions of the general anaesthetic etomidate were examined in voltage‐clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ‐aminobutyric acidA (GABAA) receptor subunits. Currents mediated by recombinant receptors with the ternary subunit composition αxβyγ2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms. For the β2‐subunit containing receptors tested, the EC50 for the potentiation of GABA‐evoked currents by etomidate (range 0.6 to 1.2 μm) was little affected by the nature of the α subunit present within the hetero‐oligomeric complex. However, replacement of the β2 by the β1 subunit produced a 9–12 fold increase in the etomidate EC50 (6 to 11 μm) for all α‐isoforms tested. For α1, α2 and α6, but not α3‐subunit containing receptors, the maximal potentiation of GABA‐evoked currents by etomidate was greater for β2‐ than for β1‐subunit containing receptors. This was most clearly exemplified by receptors composed of α6β1γ2L compared to α6β2γ2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28±2% and 169±4% of the maximal GABA response, respectively. For α1 subunit‐containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α‐pregnan‐3α‐ol‐20‐one was marginally higher for β1 rather than the β2 subunit‐containing receptor, although its maximal effect was similar at the two receptor isoforms. The GABA‐mimetic action of etomidate was supported by β2‐ but not β1‐subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β2‐subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the α isoform. It is concluded that the subtype of β‐subunit influences the potency with which etomidate potentiates GABA‐evoked currents and that the β isoform is a crucial determinant of the GABA‐mimetic activity of this compound. The nature of the α‐subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

The δ Subunit of γ-Aminobutyric Acid Type A Receptors Does Not Confer Sensitivity to Low Concentrations of Ethanol

GABAA receptors (GABAARs) are usually formed by α, β, and γ or δ subunits. Recently, δ-containing GABAARs expressed in Xenopus oocytes were found to be sensitive to low concentrations of ethanol (1–3 mM). Our objective was to replicate and extend the study of the effect of ethanol on the function of α4β3δ GABAARs. We independently conducted three studies in two systems: rat and human GABAARs expressed in Xenopus oocytes, studied through two-electrode voltage clamp; and human GABAARs stably expressed in the fibroblast L(tk–) cell line, studied through patch-clamp electrophysiology. In all cases, α4β3δ GABAARs were only sensitive to high concentrations of ethanol (100 mM in oocytes, 300 mM in the cell line). Expression of the δ subunit in oocytes was assessed through the magnitude of the maximal GABA currents and sensitivity to zinc. Of the three rat combinations studied, α4β3 was the most sensitive to ethanol, isoflurane, and 5α-pregnan-3α,21-diol-20-one (THDOC); α4β3δ and α4β3γ2S were very similar in most aspects, but α4β3δ was more sensitive to GABA, THDOC, and lanthanum than α4β3γ2S GABAARs. Ethanol at 30 mM did not affect tonic GABA-mediated currents in dentate gyrus reported to be mediated by GABAARs incorporating α4 and δ subunits. We have not been able to replicate the sensitivity of α4β3δ GABAARs to low concentrations of ethanol in four different laboratories in independent studies. This suggests that as yet unidentified factors may play a critical role in the ethanol effects on δ-containing GABAARs.

Neuroactive steroids and inhibitory neurotransmission: mechanisms of action and physiological relevance.

Dysfunction of GABA(A) receptor-mediated inhibition is implicated in a number of neurological and psychiatric conditions including epilepsy and affective disorders. Some of these conditions have been associated with abnormal levels of certain endogenously occurring neurosteroids, which potently and selectively enhance the function of the brains major inhibitory receptor, the GABA(A) receptor. Consistent with their ability to enhance neuronal inhibition, such steroids exhibit in animals and humans anxiolytic, anticonvulsant and anesthetic actions. Neurosteroids, exemplified by the potent progesterone metabolite, 5alpha-pregnan-3alpha-ol-20-one can be synthesized de novo in the CNS both in neurones and glia in levels sufficient to modulate GABA(A) receptor function. Neurosteroid levels are not static, but are subject to dynamic fluctuations, for example during stress, or the later stages of pregnancy. These observations suggest that these endogenous modulators may refine the function of the brains major inhibitory receptor and thus, play an important physiological and pathophysiological role. However, given the ubiquitous expression of GABA(A) receptors throughout the mammalian CNS, changes in neurosteroid levels should be widely experienced, causing a generalized enhancement of neuronal inhibition. Such a non-specific action would seem incompatible with a physiological role. However, neurosteroid action is both brain region and neurone selective. This specificity results from a variety of molecular mechanisms including receptor subunit composition, local steroid metabolism and phosphorylation. This paper will evaluate the relative contribution these mechanisms play in defining the interaction of neurosteroids with synaptic and extra-synaptic GABA(A) receptors.

General anaesthetic action at transmitter-gated inhibitory amino acid receptors

Research within the past decade has provided compelling evidence that anaesthetics can act directly as allosteric modulators of transmitter-gated ion channels. Recent comparative studies of the effects of general anaesthetics across a structurally homologous family of inhibitory amino acid receptors that includes mammalian GABAA, glycine and Drosophila RDL GABA receptors have provided new insights into the structural basis of anaesthetic action at transmitter-gated channels. In this article, the differential effects of general anaesthetics across inhibitory amino acid receptors and the potential relevance of such actions to general anaesthesia will be discussed.

The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study

1 The effects of five structurally dissimilar general anaesthetics were examined in voltage‐clamp recordings of agonist‐evoked currents mediated by recombinant γ‐aminobutyric acid (GABA)A receptors composed of human α1β1 and γ2L subunits expressed in Xenopus laevis oocytes. A quantitative comparison of the effects of these agents was made upon recombinant glycine receptors expressed as a homo‐oligomer of human α1 subunits, or as a hetero‐oligomer of human α1 and rat β subunits. 2 Complementary RNA‐injected oocytes expressing GABAA receptors responded to bath applied GABA with an EC50 of 158±34 μm. Oocytes expressing α1 and α1β glycine receptors subsequent to cDNA injection displayed EC50 values of 76±2 μm and 66±2 μm, respectively, in response to bath applied glycine. 3 Picrotoxin antagonized responses mediated by homo‐oligomeric α1 glycine receptors with an IC50 of 4.2±0.8 μm. Hetero‐oligomeric α1β glycine receptors were at least 100‐fold less sensitive to blockade by picrotoxin. 4 With the appropriate agonist EC10, propofol enhanced GABA and glycine‐evoked currents to approximately the maximal response produced by a saturating concentration of either agonist (i.e. Imax). The calculated EC50 values were 2.3±0.2 μm, 16±3 μm and 27±2 μm, for GABAA α1β1γ2L, glycine α1 and α1β receptors, respectively. At relatively high concentrations, propofol was observed to activate directly both GABAA and glycine receptors. 5 Pentobarbitone potentiated GABA‐evoked currents to 117±8.5% of Imax with an EC50 of 65±3 μm. The barbiturate also produced a substantial enhancement of the glycine‐evoked currents, Imax and EC50 values being 71±2% and 845±66 μm and 51±10% and 757±30 μm for homomeric α1 and heteromeric α1β glycine receptors respectively. At high concentrations, pentobarbitone directly activated GABAA, but not glycine, receptors. 6 The potentiation by propofol or pentobarbitone of currents mediated by α1 homo‐oligomeric glycine receptors was in both cases associated with a parallel sinistral shift of the glycine concentration‐effect curve. The effects of binary combinations of pentobarbitone and propofol at maximally effective concentrations were mutually occlusive suggesting a common site, or mechanism, of action. 7 GABA‐evoked currents were maximally potentiated by etomidate to 79±2% of Imax (EC50 of 8.1±0.9 μm). By contrast, glycine‐induced currents mediated by α1 and α1β glycine receptor isoforms were enhanced only to 29±4% and 28±3% of Imax. Limited solubility precluded the calculation of EC50 values for the effect of etomidate at glycine receptors. None of the receptor isoforms examined were directly activated by etomidate. 8 The neurosteroid 5α‐pregnan‐3α‐ol‐20‐one potentiated GABA‐evoked currents to 69±4% of Imax, with an EC50 value of 89±6 nm. In contrast, both α1 homo‐oligomeric and α1β heter‐oligomeric glycine receptors were insensitive to the action of this steroid. A direct agonist action of the steroid was discernible at GABAA, but not glycine, receptors. 9 Trichloroethanol, the active metabolite of the general anaesthetic chloral hydrate, enhanced glycine‐evoked currents to 77±10% and 94±4% of Imax on α1 and α1β glycine receptors, with EC50 values of 3.5±0.1 mm and 5.9±0.3 mm respectively. On GABAA receptors, trichloroethanol had a lower maximum enhancement (52±5% of Imax), but a slightly higher potency (EC50 1.0±0.1 mm). Trichloroethanol activated neither GABAA, nor glycine, receptors. 10 The data demonstrate a variety of intravenous general anaesthetic agents, at clinically relevant concentrations, to augment preferentially GABA‐evoked currents mediated by the α1β1γ2L receptor subunit combination as compared to their effects on both α1 and α1β glycine receptors. However, the presence on glycine receptors of lower affinity modulatory binding sites for pentobarbitone, propofol and trichloroethanol may aid in the identification of the molecular determinants of the CNS actions of these anaesthetics.

The Contraceptive Agent Provera Enhances GABAA Receptor-Mediated Inhibitory Neurotransmission in the Rat Hippocampus: Evidence for Endogenous Neurosteroids?

Neurosteroids typified by 5α-pregnan-3α-ol-20-one (5α3α) have emerged as the most potent endogenous positive modulators of the GABAA receptor, the principal mediator of fast inhibitory transmission within the CNS. Neurosteroids can be synthesized de novo in the brain in levels sufficient to modulate GABAA receptor function and, thus, might play an important physiological-pathophysiological role. Indirect support for this proposal comes from the observation that neurosteroid action is region and neuron selective. However, the mechanism(s) that imparts specificity of action remains primarily elusive. Although neurosteroids are relatively promiscuous toward different GABAA receptor isoforms, the contribution of local neurosteroid metabolism has been relatively unexplored. Here, we investigate the role of neurosteroid metabolism by using electrophysiological techniques to compare the actions of 5α3α and its metabolically stable synthetic analog ganaxolone on inhibitory neurotransmission in CA1 and dentate gyrus neurons. Furthermore, we evaluate the contribution of a key enzyme in neurosteroid metabolism [i.e., 3α-hydroxysteroidoxidoreductase (3α-HSOR)] to the inactivation of endogenous, or exogenously applied 5α3α. We show that low concentrations of ganaxolone, but not of 5α3α, enhance inhibitory transmission in dentate gyrus, whereas both steroids are similarly effective in CA1 neurons. Furthermore, inhibition of 3α-HSOR by the contraceptive agent Provera results in enhanced synaptic and extrasynaptic GABAA receptor-mediated inhibition in the dentate gyrus but not in the CA1 region. Collectively, these findings advocate a crucial role for local steroid metabolism in shaping GABAA receptor-mediated inhibition in a regionally dependent manner and suggest a novel action by the contraceptive agent on inhibitory centers in the CNS.

Novel compounds selectively enhance δ subunit containing GABAA receptors and increase tonic currents in thalamus

Inhibition in the brain is dominated by the neurotransmitter gamma-aminobutyric acid (GABA); operating through GABA(A) receptors. This form of neural inhibition was presumed to be mediated by synaptic receptors, however recent evidence has highlighted a previously unappreciated role for extrasynaptic GABA(A) receptors in controlling neuronal activity. Synaptic and extrasynaptic GABA(A) receptors exhibit distinct pharmacological and biophysical properties that differentially influence brain physiology and behavior. Here we used a fluorescence-based assay and cell lines expressing recombinant GABA(A) receptors to identify a novel series of benzamide compounds that selectively enhance, or activate alpha4beta3delta GABA(A) receptors (cf. alpha4beta3gamma2 and alpha1beta3gamma2). Utilising electrophysiological methods, we illustrate that one of these compounds, 4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS1) potently (low nM) enhances GABA-evoked currents mediated by alpha4beta3delta receptors. At similar concentrations DS1 directly activates this receptor and is the most potent known agonist of alpha4beta3delta receptors. 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS2) selectively potentiated GABA responses mediated by alpha4beta3delta receptors, but was not an agonist. Recent studies have revealed a tonic form of inhibition in thalamus mediated by the alpha4beta2delta extrasynaptic GABA(A) receptors that may contribute to the regulation of thalamocortical rhythmic activity associated with sleep, wakefulness, vigilance and seizure disorders. In mouse thalamic relay cells DS2 enhanced the tonic current mediated by alpha4beta2delta receptors with no effect on their synaptic GABA(A) receptors. Similarly, in mouse cerebellar granule cells DS2 potentiated the tonic current mediated by alpha6betadelta receptors. DS2 is the first selective positive allosteric modulator of delta-GABA(A) receptors and such compounds potentially offer novel therapeutic opportunities as analgesics and in the treatment of sleep disorders. Furthermore, these drugs may be valuable in elucidating the physiological and pathophysiological roles played by these extrasynaptic GABA(A) receptors.

The expression of GABAAβ subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells

The subunit composition of GABAA receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on α and γ subunits, with little attention given to β subunits. Dentate gyrus granule cells (DGGCs) express all three β subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate ‘phasic’ and ‘tonic’ transmission, respectively. To investigate the subcellular distribution of the β subunits we have utilized the patch‐clamp technique to compare the properties of ‘tonic’ and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20–26 wild‐type (WT), β2−/−, β2N265S (etomidate‐insensitive), α1−/− and δ−/− mice. Deletion of either the β2 or the δ subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the δ subunit‐preferring agonist 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the β2/3 subunit‐selective agent etomidate was significantly reduced for DGGCs derived from β2N265S mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on α4−/− mice, identify a population of extrasynaptic α4β2δ receptors, whereas synaptic GABAA receptors appear to primarily incorporate the β3 subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating α5 and γ2 subunits. Deletion of the β2 subunit had no effect on the diazepam‐induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABAA receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.