α4β3δ GABAAReceptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential
2001; Elsevier BV; Volume: 276; Issue: 42 Linguagem: Inglês
10.1074/jbc.m104318200
ISSN1083-351X
AutoresCharles Adkins, Gopalan V. Pillai, Julie Kerby, Timothy P. Bonnert, Christine Haldon, Ruth M. McKernan, Jesús González, Kahuku Oades, Paul J. Whiting, Peter B. Simpson,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoSelective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome. Selective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome. γ-aminobutyric acid γ-aminobutyric acid, type A fluorescence resonance energy transfer 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol chlorocoumarin-2-dimyristoyl phosphatidylethanolamine dimethoxy-4-ethyl-β-carboline-3-carboxylate ethyl-β-carboline-3-carboxylate γ-Aminobutyric acid (GABA)1 is the predominant inhibitory neurotransmitter in the central nervous system, and modulators of type A GABA (GABAA) receptors are used to treat anxiety, insomnia, muscle spasms, and epilepsy. GABAAreceptors are pentameric ligand-gated chloride channels, mediating rapid inhibitory synaptic neurotransmission, and are composed of different combinations of subunits from a family including α1–6, β1–4, γ1–3, δ, ε, θ, and ρ1–2 (1Whiting P.J. Bonnert T.P. McKernan R.M. Farrar S. Le-Bourdelles B. 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The association of these pathologies with changes in α4 and δ subunit expression and the observation that ligands with high affinity for α4βγ2 GABAA receptors are amethystic (35Suzdak P.D. Glowa J.R. Crawley J.N. Schwartz R.D. Skolnick P. Paul S.M. Science. 1996; 234: 1243-1247Crossref Scopus (486) Google Scholar, 36June H.L. Duemler S.E. Greene T.L. Williams J.A. Lin M. Dervaraju S.L. Chen S.H. Lewis M.J. Murphy J.M. J. Pharmacol. Exp. Ther. 1996; 274: 1105-1112Google Scholar) suggest that novel selective modulators of these GABAA receptors may, as well as leading to a better understanding of the properties and physiological roles of these subunits in the brain, have great therapeutic benefit. The development of such modulators has been held back on two counts. First, α4β3δ receptors cannot easily be expressed in transient recombinant systems, and so their properties remain unclear. Second, GABAA receptor drug-development programs have depended until now on difficult and time-consuming electrophysiological techniques or less sensitive radio-ion flux and pH methods for determining the effects of compounds on GABAAreceptor function (6Smith A.J. Alder L. Silk J. Adkins C.E. Fletcher A.E. Scales T. Kerby J. McKernan R.M. Atack J.R. Mol. Pharmacol. 2001; 59: 1108-1118Crossref PubMed Scopus (123) Google Scholar, 37Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 33: 582-585Crossref Scopus (1145) Google Scholar, 38Wafford K.A. Whiting P.J. Kemp J.A. Mol. Pharmacol. 1992; 43: 240-244Google Scholar, 39Simpson P.B. Woollacott A.J. Pillai G.V. Maubach K.A. Hadingham K.L. Martin K. Choudhury H.I. Seabrook G.R. J. Neurosci. Methods. 2000; 99: 91-100Crossref PubMed Scopus (11) Google Scholar). We have overcome these problems by creating a stable L(−tk) mouse cell line in which expression of α4β3δ receptors is under the control of a dexamethasone-induced promoter, and by developing an experimental system using fast ratiometric voltage-sensitive FRET (40Gonzalez J.E. Oades K. Leychkis Y. Harootunian A. Negulescu P.A. Drug Discov. Today. 1999; 4: 431-439Crossref PubMed Scopus (147) Google Scholar) to measure GABA-evoked changes in membrane potential. Fluorescence measurements of GABAA receptor function offer significant advantages because they are safe, are sufficiently sensitive to detect small potentiations and inhibitions, and can be miniaturized for future ultrahigh throughput applications. Furthermore, unlike high throughput radioligand binding assays, which have also been used for the development of GABAA receptor modulators, they can identify modulators regardless of their site of action. Here we describe the use of this novel fluorescence technique to characterize the pharmacological activation and modulation of GABAAreceptors with the subunit combinations α3β3γ2, α4β3γ2, and α4β3δ. L(−tk) cells were stably transfected, using a pMSGneo vector, with combinations of human GABAA receptor subunits. Expression of α, β, and γ subunits was controlled by a dexamethasone-inducible promoter as described previously (14Sur C. Farrar S. McKernan R. Atack J. Mol. Pharmacol. 1999; 56: 110-115Crossref PubMed Scopus (200) Google Scholar, 41Hadingham K.L. Wingrove P.B. Wafford K.A. Bain C. Kemp J.A. Palmer K.J. Wilson A.W. Wilcox A.S. Sikela J.M. Ragan C.I. Whiting P.J. Mol. Pharmacol. 1993; 44: 1211-1218PubMed Google Scholar), whereas expression of δ subunits was constitutive. Enzyme-linked immunosorbent assays using Myc-tagged subunits confirmed that δ subunits were only present at the cell surface if both α4 and β3 subunits were also present. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% serum (Fetalclone II) at 37 °C in an atmosphere of 5% CO2 and 95% air. Cells were passaged weekly and for experiments were transferred to 96-well black-sided microtiter plates at a density that gave confluent monolayers on the days of experiments. Receptor expression was induced 24 h before experiments by replacing 50% of the medium with medium containing dexamethasone (1 µm final concentration). All experiments were performed in a low Cl− buffer (160 mm sodium d-gluconate, 4.5 mmpotassium d-gluconate, 2 mm CaCl2, 1 mm MgCl2, 10 mmd-glucose, 10 mm HEPES, pH 7.4). Cells were washed twice, leaving a 25-µl residual volume, and 55 µl of dye solutions were added to give final concentrations of 4 µmchlorocoumarin-2-dimyristoyl phosphatidylethanolamine (CC2-DMPE) and 1 µm bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol (DisBac2)(3). After a 30-min incubation at room temperature in darkness, cells were washed again, and 65 µl of dye solutions were added to give final concentrations of 1 µmDisBac2(3) and 0.5 mm tartrazine. Microtiter plates were then placed in a voltage/ion probe reader (VIPRTM; Aurora Biosciences Corp.), which performs automated additions of pharmacological stimuli and records fluorescence emission. Briefly, the VIPRTM consists of a Hamilton 2200 pipetter, an automated microplate positioning stage, and a fiber-optic illumination and detection system capable of measuring two emission wavelengths from eight wells simultaneously (40Gonzalez J.E. Oades K. Leychkis Y. Harootunian A. Negulescu P.A. Drug Discov. Today. 1999; 4: 431-439Crossref PubMed Scopus (147) Google Scholar). A 400DF15 filter was used in the excitation pathway, and 460DF45 and 580DF60 filters were used in the respective emission pathways. In all experiments, basal fluorescence was read for 8 s before addition of modulators, and then GABA was added 22 s later. Fluorescence emission from wells was recorded at 1 Hz. For each time point and for each fluorescence emission wavelength, we subtracted background fluorescence recorded from wells without cells in the same microtiter plate and calculated the ratio of fluorescence at 460 nm to that at 580 nm. GABA-evoked depolarizations were then expressed as a fractional change in this ratio. Algorithms written as Excel 97 (Microsoft Corp.) macros were used for automated calculations of fluorescence ratio and GABA responses (39Simpson P.B. Woollacott A.J. Pillai G.V. Maubach K.A. Hadingham K.L. Martin K. Choudhury H.I. Seabrook G.R. J. Neurosci. Methods. 2000; 99: 91-100Crossref PubMed Scopus (11) Google Scholar), and an iterative curve-fitting program (Prism, GraphPad Software Inc.) was used to fit concentration-effect relationships to a four-parameter logistic equation. DisBac2(3) and CC2-DMPE were from Aurora Biosciences Corp. Dulbecco's modified Eagle's medium was from Life Technologies, Inc., and Fetalclone II was from Hyclone (Logan, UT). Loreclezole was a gift from Janssen, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) was from Tocris (Baldwin, MO), and bretazenil was synthesized by Merck Sharp & Dohme Research Laboratories. Tartrazine, gluconate salts, and all other GABAA receptor modulators were obtained from Sigma. All other reagents were of the highest analytical grade available. Previously, optical sensors of membrane potential operated through a slow redistribution of permeant ions or a rapid but insensitive perturbation of dyes attached to one face of the membrane (42Waggoner A.S. Grinvald F. Ann. N. Y. Acad. Sci. 1977; 303: 217-241PubMed Google Scholar, 43Loew L.M. Spectroscopic Membrane Probes. CRC Press, Boca Raton, FL1988: 139-151Google Scholar, 44Loew L.M. Fluorescent and Luminescent Probes for Biological Activity. WAcademic, San Diego1993: 150-160Google Scholar). However, a recently developed membrane potential indicator, described in Fig. 1, uses FRET to provide a fluorescent readout of membrane potential that is both rapid and robust (45Gonzalez J.E. Tsien R.Y. Chem. Biol. 1997; 4: 269-277Abstract Full Text PDF PubMed Scopus (172) Google Scholar). Before using this technique to characterize cell lines expressing α4 subunit-containing GABAA receptors, we first established its pharmacological utility using cells expressing the well characterized subunit combination α3β3γ2. In low chloride medium, GABA-evoked depolarizations of cells expressing α3β3γ2 GABAAreceptors and loaded with CC2-DMPE and DisBac2(3) were rapidly transduced into decreased FRET, and, therefore, an increase in the ratio of fluorescence emission at 460 nm to that at 580 nm was seen (Fig. 2). The fluorescence emission ratio rose to a concentration-dependent plateau within 5 s that was sustained for >15 s. For the plateau phase of the response, measured as the mean normalized fluorescence emission ratio between 10 and 15 s after application of agonist, the half-maximal concentration (EC50) of GABA was 2.1 ± 0.2 µm, and the Hill slope (nH) was 1.5 ± 0.1 (mean ± S.E. of three experiments, Fig. 2b). We next examined receptor pharmacology by pretreating cells with compounds known to be active at α3β3γ2GABAA receptors and then applying a half-maximal concentration of GABA. α3β3γ2responses were blocked by the antagonists bicuculline (competitive) and picrotoxin (noncompetitive), potentiated by the benzodiazepine agonist zolpidem, and partially inhibited by the benzodiazepine inverse agonist dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) (Fig.2c). These findings are highly consistent with those from electrophysiological experiments (38Wafford K.A. Whiting P.J. Kemp J.A. Mol. Pharmacol. 1992; 43: 240-244Google Scholar).Figure 2FRET-derived measurements of depolarizations mediated by α3β3γ2 GABAA receptors.a, L(−tk) cells expressing α3β3γ2GABAA receptors and loaded with the membrane potential indicator dye pair were stimulated with 30 (▪), 10 (●), 6 (▴), 3 (▾), 2 (♦), 1 (○), or 0.1 (⋄) µm GABA (shown by the black bar). The response shown is the ratio of fluorescence at 460 nm (f460) to that at 580 nm (f580) normalized to the mean ratio over the first 5 s of recording. b, the plateau ratio from a, taken as the mean between 10 and 15 s after addition of GABA, is shown as a function of GABA concentration. c, cells were pretreated with different concentrations of picrotoxin (●), bicuculline (▾), DMCM (○), or zolpidem (▴) before addition of GABA at a previously established half-maximal concentration (EC50). The effect of these compounds on GABA responses (calculated as for b) is shown as the percentage difference from control wells where no modulator was added. Maximum effects and EC50 values for these compounds were as follows: picrotoxin, −85% and 6.7 µm; bicuculline, −86% and 3.5 µm; DMCM, −28% and 0.044 µm; zolpidem, +32% and 0.055 µm. The data shown are the mean ± S.E. of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Having established that fluorescence measurements of GABAAreceptor function appear to reliably report receptor pharmacology, we then examined GABA-evoked changes in FRET using L(−tk) cells expressing either α4β3γ2GABAA receptors or the previously uncharacterized subunit combination α4β3δ. The kinetics of GABA-evoked depolarization were similar for these cells to those for cells expressing α3β3γ2GABAA receptors (Fig.3a). GABA, muscimol, and THIP were between 3 and 6 times more potent at α4β3δ receptors compared with α4β3γ2 (Figs. 2band 3b). The first detectable response to muscimol occurred 1 s earlier than that for GABA or THIP, but thereafter the three agonists evoked changes in fluorescence ratio with similar kinetics (Fig. 3c). Although less potent than GABA, THIP was a fully efficacious agonist at α4β3γ2receptors and a superagonist at α4β3δ (Fig. 3b). Responses mediated by both α4β3δ and α4β3γ2 receptors were inhibited by pretreatment with picrotoxin and bicuculline. Whereas picrotoxin (30 µm) inhibited the responses to all concentrations of GABA, bicuculline (30 µm) inhibited only submaximal responses, causing a 30-fold shift in the GABA concentration-response curve (Fig. 3d). We then examined the regulation of α4 subunit-containing GABAA receptors by a variety of known modulators of GABAA receptors, including benzodiazepines, steroids, and anesthetics (Table I). α4β3γ2 receptor-mediated responses were partially inhibited by pretreatment with DMCM, which had a similar efficacy to that at α3β3γ2.α4β3γ2responses were potentiated by nanomolar concentrations of bretazenil and Ro15-4513 but were insensitive to the classical benzodiazepine site agonists zolpidem and flunitrazepam (Fig.4). α4β3δ receptors were largely insensitive to the benzodiazepine site modulators used in this study with just two exceptions. First, Ro15-4513 inhibited α4β3δ-mediated responses, although with an EC50 100 times higher than that for its potentiation of α4β3γ2 receptors. Second, micromolar concentrations of ethyl-β-carboline-3-carboxylate (β-CCE) potentiated responses mediated by both α4β3γ2 and α4β3δ receptors (Fig. 4). Micromolar concentrations of furosemide, an inhibitor of GABAAreceptors (20Wafford K.A. Thompson S.A. Thomas D. Sikela J. Wilcox A.S. Whiting P.J. Mol. Pharmacol. 1996; 50: 670-678PubMed Google Scholar, 46Korpi E.R. Lüddens H. Br. J. Pharmacol. 1997; 120: 741-748Crossref PubMed Scopus (68) Google Scholar), selectively inhibited α4β3γ2 receptors with no discernible effect on α4β3δ (TableI).Table IModulation of α4β3γ2 and α4β3δ GABAA receptorsα4β3γ2α4β3δEfficacyEC50EfficacyEC50%µm%µmPicrotoxin−909.6−1114.7Bicuculline−868.7−924.3DMCM−280.068NoneBretazenil+320.12NoneRo15-4513+210.052−434.9ZolpidemNoneNoneFlunitrazepamNoneNoneβ-CCE+214.5+376.5β-CCMNoneNoneFurosemide−27>3NonePropofol+341.0+39>3Loreclezole+3311+3118Pregnenolone−955.8−1003.9Pregnanolone−2714−899.7Alphaxalone+320.11+1200.12Pentobarbital+1691+110>300L(−tk) cells expressing α4β3γ2 or α4β3δ receptors were pretreated with modulators of GABAA receptors before addition of GABA at half-maximal concentration. The effects of modulators on GABA responses (calculated as for Fig. 1b) were calculated as the percentage difference from the agonist response in parallel wells in which no modulator was added. The maximum effects shown are the mean of three experiments. β-CCM, methyl-β-carboline-3-carboxylate. Open table in a new tab L(−tk) cells expressing α4β3γ2 or α4β3δ receptors were pretreated with modulators of GABAA receptors before addition of GABA at half-maximal concentration. The effects of modulators on GABA responses (calculated as for Fig. 1b) were calculated as the percentage difference from the agonist response in parallel wells in which no modulator was added. The maximum effects shown are the mean of three experiments. β-CCM, methyl-β-carboline-3-carboxylate. α4β3γ2 and α4β3δ responses were potentiated by the anesthetics propofol and loreclezole and inhibited by the steroid pregnenolone (5-pregnen-3β-ol-20-one). These compounds were of similar potency and efficacy at the two receptor types (Fig.5 and Table I). The steroid pregnanolone (5β-pregnan-3α-ol-20-one) inhibited, and alphaxalone (5α-pregnan-3α-ol-11,20-dione) potentiated responses at both γ2- and δ-containing receptors. These agents had 3–4 times greater efficacy at α4β3δ compared with α4β3γ2 (Fig. 5). Alphaxalone, at concentrations above 3 µm, also directly activated GABAA receptors, evoking a depolarization of both cell types, again with greater efficacy at α4β3δ receptors (Fig. 5c). In contrast, applications of pregnenolone and pregnanolone, at concentrations of up to 30 µm, did not affect membrane potential directly. The barbiturate pentobarbital was another more efficacious (7-fold) potentiator of α4β3δ receptors compared with α4β3γ2 (Fig. 5b). As well as potentiating GABA responses, barbiturates can directly activate GABAA receptors, and this effect of pentobarbital showed the reverse subtype selectivity. Whereas high concentrations of pentobarbital depolarized cells expressing α4β3γ2 receptors, there was no discernible effect at α4β3δ (Fig.5c). In this study we have developed a novel fluorescence technique that provides rapid and sensitive measurements of GABAAreceptor function, and have used it to characterize a novel cell line expressing GABAA receptors with the composition α4β3δ. Our initial experiments, using cell lines expressing the previously characterized GABAAreceptor subunit combinations α3β3γ2 and α4β3γ2, demonstrated that GABAA receptor-mediated chloride fluxes were rapidly and reliably transduced into decreased FRET. In contrast to traditional fluorescence assays of membrane potential utilizing oxonol redistribution, GABA-evoked depolarization of cells loaded with CC2-DMPE and DisBac2(3) and excited with 410 nm light leads to a change in fluorescence emission that occurs within seconds rather than minutes. As previously reported, substitution of α4subunits for α3 did not affect GABA potency, which was similar to that previously reported for the same subunit combinations expressed in mammalian cells (6Smith A.J. Alder L. Silk J. Adkins C.E. Fletcher A.E. Scales T. Kerby J. McKernan R.M. Atack J.R. Mol. Pharmacol. 2001; 59: 1108-1118Crossref PubMed Scopus (123) Google Scholar, 19Knoflach F. Benke D. Wang Y. Scheurer L. Hartmut L. Hamilton B.J. Carter D.B. Mohler H. Benson J.A. Mol. Pharmacol. 1996; 50: 1253-1261PubMed Google Scholar). GABA-evoked responses were blocked by picrotoxin and bicuculline, and at α3β3γ2 and α4β3γ2 receptors, the efficacies and potencies of the benzodiazepines tested were very similar to published values (6Smith A.J. Alder L. Silk J. Adkins C.E. Fletcher A.E. Scales T. Kerby J. McKernan R.M. Atack J.R. Mol. Pharmacol. 2001; 59: 1108-1118Crossref PubMed Scopus (123) Google Scholar, 19Knoflach F. Benke D. Wang Y. Scheurer L. Hartmut L. Hamilton B.J. Carter D.B. Mohler H. Benson J.A. Mol. Pharmacol. 1996; 50: 1253-1261PubMed Google Scholar, 20Wafford K.A. Thompson S.A. Thomas D. Sikela J. Wilcox A.S. Whiting P.J. Mol. Pharmacol. 1996; 50: 670-678PubMed Google Scholar, 38Wafford K.A. Whiting P.J. Kemp J.A. Mol. Pharmacol. 1992; 43: 240-244Google Scholar, 47Whittemore E.R. Yang W. Drewe J.A. Woodward R.M. Mol. Pharmacol. 1996; 50: 1364-1375PubMed Google Scholar). Thus FRET-derived measurements of membrane potential proved to be a sensitive and reliable indicator of GABAA receptor pharmacology. They gave results that were essentially indistinguishable from those for electrophysiological experiments and are likely to prove useful for studies of multiple receptor classes. While GABAA receptors composed of α, β, and γ subunits have been studied extensively, relatively little is known about the functional and pharmacological properties of receptor isoforms containing δ subunits. Receptors containing δ subunits in combination with α4, as occur in situ, have never been characterized. We therefore created a novel L(−tk) cell line in which expression of α4β3δ GABAA receptors was under the control of a dexamethasone-inducible promoter, and used FRET-derived measurements of membrane potential to directly compare them to α4β3γ2 receptors. We found that δ subunits, compared with γ2, conferred higher affinity for all the agonists tested. The rank order for agonist potency muscimol > GABA > THIP was unchanged, but THIP acted as a superagonist at α4β3δ receptors, evoking substantially larger changes in FRET than either GABA or muscimol. Partial agonists at other GABAA receptor subtypes have been described, but no agonist has shown greater efficacy than GABA. An equally valid interpretation of this data, therefore, is that δ subunits, when combined with α4 and β3, confer partial agonism to GABA. The different potency, and perhaps efficacy, of GABA at α4β3γ2 and α4β3δ receptors suggest quite different physiological roles for these receptor isoforms. Low affinity receptors containing γ2 subunits may be suited to synapses where GABA is plentiful and a rapid dissociation rate is beneficial to high frequency signaling. Higher affinity δ subunit-containing receptors may, in an extrasynaptic location where GABA is at lower concentrations (48Nusser Z. Sieghart W. Somogyi P. J. Neurosci. 1998; 18: 1693-1703Crossref PubMed Google Scholar, 49Brickley S.G. Cull-Candy S.G. Farrant M. J. Neurosci. 1999; 19: 2960-2973Crossref PubMed Google Scholar), have a modulatory role for which rapid responses are not required and for which a lower conductance is more appropriate. δ subunits were an important determinant of the effects of a variety of allosteric modulators, including benzodiazepines, steroids, and barbiturates. Substitution of γ2 subunits with δ abolished sensitivity to modulators acting at the benzodiazepine binding site. Although β-carbolines, such as β-CCE and methyl-β-carboline-3-carboxylate, inhibit GABAA receptors with high potency via the benzodiazepine binding site, they also potentiate GABA responses, with lower potency, via the loreclezole site present only on β2- and β3-containing receptors (10Stevenson A. Wingrove P.B. Whiting P.J. Wafford K.A. Mol. Pharmacol. 1995; 48: 965-969PubMed Google Scholar). Therefore the potentiation of both α4β3γ2and α4β3δ responses by β-CCE was almost certainly mediated by the binding site for loreclezole and does not indicate benzodiazepine sensitivity. Barbiturates are thought to potentiate the response of GABAA receptors irrespective of their subunit composition (47Whittemore E.R. Yang W. Drewe J.A. Woodward R.M. Mol. Pharmacol. 1996; 50: 1364-1375PubMed Google Scholar). Both α4β3γ2 and α4β3δ responses were potentiated by pentobarbital. However, δ subunits, compared with γ2, conferred 7 times higher efficacy to pentobarbital. At micromolar concentrations, barbiturates have a second effect, directly activating GABAA receptors (50MacDonald R. Barker L. Nature. 1978; 271: 563-564Crossref PubMed Scopus (221) Google Scholar, 51Borman J. Trends Neurosci. 1988; 11: 112-116Abstract Full Text PDF PubMed Scopus (566) Google Scholar). α4β3γ2 receptors were activated by pentobarbital, but this effect was abolished when γ2 subunits were substituted with δ. We conclude that δ and γ2 subunits affect both the modulation and activation of GABAA receptors by barbiturates. There may also be a role for β subunits since α4β3γ2 and α4β2γ2 receptors are activated by pentobarbital (47Whittemore E.R. Yang W. Drewe J.A. Woodward R.M. Mol. Pharmacol. 1996; 50: 1364-1375PubMed Google Scholar), whereas the effect does not occur on α4β1γ2 (20Wafford K.A. Thompson S.A. Thomas D. Sikela J. Wilcox A.S. Whiting P.J. Mol. Pharmacol. 1996; 50: 670-678PubMed Google Scholar). α4β3γ2 and α4β3δ receptors were differentially modulated by steroids. In contrast to the stimulatory effect at other GABAA receptors (52Hevers W. Lüddens H. Mol. Neurobiol. 1998; 18: 35-86Crossref PubMed Scopus (410) Google Scholar), receptors containing α4subunits were inhibited by the naturally occurring neurosteroid pregnanolone. Furthermore, both pregnanolone and the synthetic anesthetic alphaxalone (52Hevers W. Lüddens H. Mol. Neurobiol. 1998; 18: 35-86Crossref PubMed Scopus (410) Google Scholar, 53Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. Ther. 1989; 248: 960-966PubMed Google Scholar) were more efficacious at α4β3δ compared with α4β3γ2. These data demonstrate that δ subunits are a critical determinant of neurosteroid efficacy, possibly accounting for the reduced behavioral effects of alphaxalone and pregnanolone in mice lacking δ subunits (55Mihalek R.M. Bannerjee P.K. Korpi E.R. Quinlan J.J. Firestone L.L. Mi Z.-P. Lagenaur C. Tretter V. Sieghart W. Anagnostaras S.G. Sage J.R. Fanselow M.S. Guidotti A. Spigelman I. Li Z. DeLorey T.M. Olsen R.W. Homanics G.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12905-12910Crossref PubMed Scopus (446) Google Scholar). During the menstrual cycle and pregnancy in normal women, levels of pregnanolone correlate with those of progesterone from which it is synthesized (54Paul S.M. Purdy R.H. FASEB J. 1992; 6: 2311-2322Crossref PubMed Scopus (1463) Google Scholar). In addition to their effects on α4subunit expression (30Moran M.H. Smith S.S. Brain Res. 1998; 807: 84-90Crossref PubMed Scopus (99) Google Scholar, 31Smith S.S. Gong Q.H. Hsu F.-C. Markowitz R.S. French-Mullen J.M.H. Li X. Nature. 1998; 392: 926-930Crossref PubMed Scopus (519) Google Scholar, 32Smith S.S. Gong Q.H. Li X. Moran M.H. Bitran D. Frye C.A. Hsu F.-C. J. Neurosci. 1998; 18: 5275-5284Crossref PubMed Google Scholar, 33Grobin A.C. Morrow A.L. Eur. J. Pharmacol. 2000; 409: R1-R2Crossref PubMed Scopus (30) Google Scholar, 34Follesa P. Serra M. Cagetti E. Pisu M.G. Porta S. Floris S. Massa F. Sanna E. Biggio G. Mol. Pharmacol. 2000; 51: 1262-1270Google Scholar), endogenous neuroactive steroids may therefore also modulate the function of GABAA receptors, particularly those containing δ subunits, and thereby contribute to the increased incidence of anxiety and seizures in premenstrual syndrome and postpartum and postmenopausal dysphoria. Our data imply that α4β3δ receptors may have a central role in these disorders and that new therapies might be developed by selective targeting of the steroid binding site of GABAAreceptors containing δ subunits. FRET-derived measurements of membrane potential provide the most robust and reliable high throughput assay of GABAA receptor function yet developed and will be an invaluable tool for characterizing novel subunit combinations and identifying new therapeutic modulators. When applied to cell lines expressing GABAA receptors with the subunit combinations α4β3γ2 and α4β3δ, this novel fluorescence technique revealed that δ subunits are an important determinant of the efficacy and potency of agonists and allosteric modulators. Of particular importance was the finding that α4β3δ receptors were markedly more sensitive to inhibition by pregnanolone, suggesting that this receptor subtype could be targeted for the treatment of premenstrual syndrome. We thank Graham Foster for developing data analysis software for fluorescence experiments and Research Information Management at Merck Sharp & Dohme for help in preparing Fig. 1.
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