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J. Biol. Chem., Vol. 276, Issue 42, 38934-38939, October 19, 2001

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₄₃ GABA_A Receptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential^*

Charles E. Adkins, Gopalan V. Pillai, Julie Kerby, Timothy P. Bonnert, Christine Haldon, Ruth M. McKernan, Jesus E. Gonzalez^§, Kahuku Oades^§, Paul J. Whiting, and Peter B. Simpson

From the Neuroscience Research Centre, Terlings Park, Merck Sharp & Dohme, Eastwick Road, Harlow, Essex, United Kingdom CM20 2QR and ^§ Aurora Biosciences Corp., La Jolla, California 92121

Received for publication, May 11, 2001, and in revised form, August 5, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Selective modulators of -aminobutyric acid, type A (GABA_A) receptors containing ₄ subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(tk) cells, we stably expressed the native GABA_A receptor subunit combinations _{332, 432}, and, for the first time, ₄₃ 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 GABA_A receptor antagonists and, for ₃₃₂ and ₄₃₂ receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with ₄ and ₃,  subunits, compared with ₂, 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. ₄₃ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at ₄₃ compared with _432. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA_A receptor. For GABA_A receptors containing ₄ subunits, our experiments reveal that inclusion of  instead of ₂ subunits 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 ₄₃ receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

-Aminobutyric acid (GABA)¹ is the predominant inhibitory neurotransmitter in the central nervous system, and modulators of type A GABA (GABA_A) receptors are used to treat anxiety, insomnia, muscle spasms, and epilepsy. GABA_A receptors are pentameric ligand-gated chloride channels, mediating rapid inhibitory synaptic neurotransmission, and are composed of different combinations of subunits from a family including _1-6, _1-4, _1-3, , , , and _1-2 (1). They are modulated by a plethora of clinically important drugs including benzodiazepines, barbiturates, steroids, and anesthetics. Subunit stoichiometry has been contentious (2-4), but the evidence is now convincing that receptors composed of , , and  subunits contain two , two , and one  subunit (5). The precise combination of subunits is an important determinant of receptor pharmacology;  subunits govern GABA affinity (6),  and  subunits regulate benzodiazepine site pharmacology (6-9), and  subunits control loreclezole and etomidate sensitivity (10).

₄ subunits comprise only a small percentage of neuronal subunits, concentrated in hippocampus, striatum, cerebral cortex, thalamus, and basal ganglia (11-15). They assemble with _2/3 and ₂ subunits in most areas of the brain (12), but also with _2/3 and  subunits in olfactory bulb, dentate gyrus, and thalamus (14-17). Of the 20-27% of thalamic GABA_A receptors that contain ₄ subunits, approximately one-third contain ₂ subunits, and two-thirds contain  subunits (14). Compared with other GABA_A receptors, those containing ₄ subunits differ in their rectification properties (18), affinity for GABA (19), and modulation by benzodiazepines (20). Receptors containing ₄ and  subunits lack benzodiazepine binding sites entirely, and those containing ₄, , and ₂ subunits have a benzodiazepine binding site that is atypical (6, 14, 21).

A variety of animal models of epilepsy lead to changes in the level of expression of ₄ and  subunit protein and mRNA in hippocampal dentate gyrus (17, 22-26) and thalamic relay nuclei (27), and acute pentylenetetrazol-induced seizures, to which mice lacking  subunits are more susceptible (56), lead to an increase in  subunit expression in neocortex (28). Elevated levels of ₄ subunits are also implicated in an animal model of alcohol dependence (29) and in steroid-withdrawal models of premenstrual syndrome and postpartum or postmenopausal dysphoria, particularly the increased anxiety and incidence of seizures (30-34). The association of these pathologies with changes in ₄ and  subunit expression and the observation that ligands with high affinity for ₄₂ GABA_A receptors are amethystic (35, 36) suggest that novel selective modulators of these GABA_A 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, ₄₃ receptors cannot easily be expressed in transient recombinant systems, and so their properties remain unclear. Second, GABA_A 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 GABA_A receptor function (6, 37-39). We have overcome these problems by creating a stable L(tk) mouse cell line in which expression of ₄₃ receptors is under the control of a dexamethasone-induced promoter, and by developing an experimental system using fast ratiometric voltage-sensitive FRET (40) to measure GABA-evoked changes in membrane potential. Fluorescence measurements of GABA_A 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 GABA_A 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 GABA_A receptors with the subunit combinations ₃₃₂, ₄₃₂, and ₄₃.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of ₃₃₂, ₄₃₂, and ₄₃ GABA_A Receptors-- L(tk) cells were stably transfected, using a pMSGneo vector, with combinations of human GABA_A receptor subunits. Expression of , , and  subunits was controlled by a dexamethasone-inducible promoter as described previously (14, 41), whereas expression of  subunits was constitutive. Enzyme-linked immunosorbent assays using Myc-tagged subunits confirmed that  subunits were only present at the cell surface if both ₄ and ₃ 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).

Fluorescence Measurements of Membrane Potential-- All experiments were performed in a low Cl buffer (160 mM sodium D-gluconate, 4.5 mM potassium D-gluconate, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM D-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 µM chlorocoumarin-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 µM DisBac₂(3) and 0.5 mM tartrazine. Microtiter plates were then placed in a voltage/ion probe reader (VIPR^TM; Aurora Biosciences Corp.), which performs automated additions of pharmacological stimuli and records fluorescence emission. Briefly, the VIPR^TM 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 (40). 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.

Data Analysis-- 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 (39), and an iterative curve-fitting program (Prism, GraphPad Software Inc.) was used to fit concentration-effect relationships to a four-parameter logistic equation.

Materials and Methods-- 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 GABA_A receptor modulators were obtained from Sigma. All other reagents were of the highest analytical grade available.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (42-44). 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 (45). Before using this technique to characterize cell lines expressing ₄ subunit-containing GABA_A receptors, we first established its pharmacological utility using cells expressing the well characterized subunit combination ₃₃₂. In low chloride medium, GABA-evoked depolarizations of cells expressing ₃₃₂ GABA_A receptors 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 (n_H) 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 ₃₃₂ GABA_A receptors and then applying a half-maximal concentration of GABA. ₃₃₂ responses 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--carboline-3-carboxylate (DMCM) (Fig. 2c). These findings are highly consistent with those from electrophysiological experiments (38).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   FRET-derived measurements of GABA-evoked depolarizations. The voltage sensor probe is composed of two fluorescent components. DisBac2(3) (shown as a red sphere) is a highly fluorescent hydrophobic anion that rapidly redistributes, in a Nernstian manner, between two energy minima on opposite sides of the plasma membrane. CC2-DMPE (shown as a blue sphere) binds specifically to the outer face of the plasma membrane and functions as a FRET donor to DisBac2(3) (a). When cells are bathed in low Cl buffer, activation of GABAA receptors (b) leads to Cl efflux and depolarizes the cell membrane. DisBac2(3) establishes a new equilibrium, and this voltage-dependent redistribution is transduced into decreased FRET efficiency and, therefore, a decrease in the ratio of fluorescence emission at 460 nm to that at 580 nm.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   FRET-derived measurements of depolarizations mediated by 332 GABAA receptors. a, L(tk) cells expressing 332 GABAA 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.

Having established that fluorescence measurements of GABA_A receptor function appear to reliably report receptor pharmacology, we then examined GABA-evoked changes in FRET using L(tk) cells expressing either ₄₃₂ GABA_A receptors or the previously uncharacterized subunit combination ₄₃. The kinetics of GABA-evoked depolarization were similar for these cells to those for cells expressing ₃₃₂ GABA_A receptors (Fig. 3a). GABA, muscimol, and THIP were between 3 and 6 times more potent at ₄₃ receptors compared with ₄₃₂ (Figs. 2b and 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 ₄₃₂ receptors and a superagonist at ₄₃ (Fig. 3b). Responses mediated by both ₄₃ and ₄₃₂ 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).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Activation of 332, 432, and 43 GABAA receptors by GABA site agonists. a, L(tk) cells expressing 332 (black), 432 (blue), or 43 (pink) GABAA receptors were stimulated with half-maximal concentrations of GABA (shown by the red bar). b, the plateau of agonist-evoked changes in fluorescence ratio (calculated as in Fig. 1b and normalized with respect to maximum GABA) is shown for cells expressing 432 (squares) or 43 (circles) GABAA receptors stimulated with different concentrations of GABA (black), muscimol (blue), or THIP (pink). c, cells expressing 432 GABAA receptors were simulated with maximal concentrations of GABA (black), muscimol (blue), or THIP (pink). d, cells expressing 43 receptors were pretreated with picrotoxin (30 µM, blue) or bicuculline (30 µM, pink) before addition of different concentrations of GABA. Responses from untreated cells measured in parallel are also shown (black). All data are the mean ± S.E. of three experiments. f460, fluorescence at 460 nm; f580, fluorescence at 580 nm.

We then examined the regulation of ₄ subunit-containing GABA_A receptors by a variety of known modulators of GABA_A receptors, including benzodiazepines, steroids, and anesthetics (Table I). ₄₃₂ receptor-mediated responses were partially inhibited by pretreatment with DMCM, which had a similar efficacy to that at ₃₃₂.₄₃₂ responses were potentiated by nanomolar concentrations of bretazenil and Ro15-4513 but were insensitive to the classical benzodiazepine site agonists zolpidem and flunitrazepam (Fig. 4). ₄₃ receptors were largely insensitive to the benzodiazepine site modulators used in this study with just two exceptions. First, Ro15-4513 inhibited ₄₃-mediated responses, although with an EC₅₀ 100 times higher than that for its potentiation of ₄₃₂ receptors. Second, micromolar concentrations of ethyl--carboline-3-carboxylate (-CCE) potentiated responses mediated by both ₄₃₂ and ₄₃ receptors (Fig. 4). Micromolar concentrations of furosemide, an inhibitor of GABA_A receptors (20, 46), selectively inhibited ₄₃₂ receptors with no discernible effect on ₄₃ (Table I).

View this table: [in this window] [in a new window]   Table I Modulation of 432 and 43 GABAA receptors L(tk) cells expressing 432 or 43 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. -CCM, methyl--carboline-3-carboxylate.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Modulation of 432 GABAA receptors by benzodiazepines. L(tk) cells expressing 432 GABAA receptors were pretreated with different concentrations of DMCM (), bretazenil (), or -CCE () before addition of GABA at half-maximal concentration. The effects of these compounds were calculated as in Fig. 1. Data shown are the mean ± S.E. of three experiments.

₄₃₂ and ₄₃ responses were potentiated by the anesthetics propofol and loreclezole and inhibited by the steroid pregnenolone (5-pregnen-3-ol-20-one). These compounds were of similar potency and efficacy at the two receptor types (Fig. 5 and Table I). The steroid pregnanolone (5-pregnan-3-ol-20-one) inhibited, and alphaxalone (5-pregnan-3-ol-11,20-dione) potentiated responses at both ₂- and -containing receptors. These agents had 3-4 times greater efficacy at ₄₃ compared with ₄₃₂ (Fig. 5). Alphaxalone, at concentrations above 3 µM, also directly activated GABA_A receptors, evoking a depolarization of both cell types, again with greater efficacy at ₄₃ 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 ₄₃ receptors compared with ₄₃₂ (Fig. 5b). As well as potentiating GABA responses, barbiturates can directly activate GABA_A receptors, and this effect of pentobarbital showed the reverse subtype selectivity. Whereas high concentrations of pentobarbital depolarized cells expressing ₄₃₂ receptors, there was no discernible effect at ₄₃ (Fig. 5c).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Modulation and activation of 432 and 43 GABAA receptors by steroids and anesthetics. L(tk) cells expressing 432 (squares) or 43 (circles) GABAA receptors were pretreated with different concentrations of alphaxalone (a, open), pregnenolone (a, filled), pregnanolone (b, filled), or pentobarbital (b, open) before application of GABA at half-maximal concentration. c, subtype-selective activation of GABAA receptors by alphaxalone and pentobarbital. L(tk) cells expressing 432 (squares) or 43 (circles) GABAA receptors were stimulated with alphaxalone (10 µM, filled) or pentobarbital (1 mM, open), addition of which is shown by the black bar. Responses were normalized with respect to maximum GABA as in Fig. 3. All data are the mean ± S.E. of three experiments. At high concentrations, pregnenolone and pregnanolone interfered with fluorescence emission, and their effects could not be interpreted at concentrations above 30 µM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we have developed a novel fluorescence technique that provides rapid and sensitive measurements of GABA_A receptor function, and have used it to characterize a novel cell line expressing GABA_A receptors with the composition ₄₃. Our initial experiments, using cell lines expressing the previously characterized GABA_A receptor subunit combinations ₃₃₂ and ₄₃₂, demonstrated that GABA_A 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 ₄ subunits for ₃ did not affect GABA potency, which was similar to that previously reported for the same subunit combinations expressed in mammalian cells (6, 19). GABA-evoked responses were blocked by picrotoxin and bicuculline, and at ₃₃₂ and ₄₃₂ receptors, the efficacies and potencies of the benzodiazepines tested were very similar to published values (6, 19, 20, 38, 47). Thus FRET-derived measurements of membrane potential proved to be a sensitive and reliable indicator of GABA_A 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 GABA_A receptors composed of , , and  subunits have been studied extensively, relatively little is known about the functional and pharmacological properties of receptor isoforms containing  subunits. Receptors containing  subunits in combination with ₄, as occur in situ, have never been characterized. We therefore created a novel L(tk) cell line in which expression of ₄₃ GABA_A receptors was under the control of a dexamethasone-inducible promoter, and used FRET-derived measurements of membrane potential to directly compare them to ₄₃₂ receptors. We found that  subunits, compared with ₂, 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 ₄₃ receptors, evoking substantially larger changes in FRET than either GABA or muscimol. Partial agonists at other GABA_A receptor subtypes have been described, but no agonist has shown greater efficacy than GABA. An equally valid interpretation of this data, therefore, is that  subunits, when combined with ₄ and ₃, confer partial agonism to GABA. The different potency, and perhaps efficacy, of GABA at ₄₃₂ and ₄₃ receptors suggest quite different physiological roles for these receptor isoforms. Low affinity receptors containing ₂ subunits may be suited to synapses where GABA is plentiful and a rapid dissociation rate is beneficial to high frequency signaling. Higher affinity  subunit-containing receptors may, in an extrasynaptic location where GABA is at lower concentrations (48, 49), have a modulatory role for which rapid responses are not required and for which a lower conductance is more appropriate.

subunits were an important determinant of the effects of a variety of allosteric modulators, including benzodiazepines, steroids, and barbiturates. Substitution of ₂ subunits with  abolished sensitivity to modulators acting at the benzodiazepine binding site. Although -carbolines, such as -CCE and methyl--carboline-3-carboxylate, inhibit GABA_A receptors with high potency via the benzodiazepine binding site, they also potentiate GABA responses, with lower potency, via the loreclezole site present only on ₂- and ₃-containing receptors (10). Therefore the potentiation of both ₄₃₂ and ₄₃ responses by -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 GABA_A receptors irrespective of their subunit composition (47). Both ₄₃₂ and ₄₃ responses were potentiated by pentobarbital. However,  subunits, compared with ₂, conferred 7 times higher efficacy to pentobarbital. At micromolar concentrations, barbiturates have a second effect, directly activating GABA_A receptors (50, 51). ₄₃₂ receptors were activated by pentobarbital, but this effect was abolished when ₂ subunits were substituted with . We conclude that  and ₂ subunits affect both the modulation and activation of GABA_A receptors by barbiturates. There may also be a role for  subunits since ₄₃₂ and ₄₂₂ receptors are activated by pentobarbital (47), whereas the effect does not occur on ₄₁₂ (20).

₄₃₂ and ₄₃ receptors were differentially modulated by steroids. In contrast to the stimulatory effect at other GABA_A receptors (52), receptors containing ₄ subunits were inhibited by the naturally occurring neurosteroid pregnanolone. Furthermore, both pregnanolone and the synthetic anesthetic alphaxalone (52, 53) were more efficacious at ₄₃ compared with ₄₃₂. These data demonstrate that  subunits are a critical determinant of neurosteroid efficacy, possibly accounting for the reduced behavioral effects of alphaxalone and pregnanolone in mice lacking  subunits (55). During the menstrual cycle and pregnancy in normal women, levels of pregnanolone correlate with those of progesterone from which it is synthesized (54). In addition to their effects on ₄ subunit expression (30-34), endogenous neuroactive steroids may therefore also modulate the function of GABA_A receptors, particularly those containing  subunits, and thereby contribute to the increased incidence of anxiety and seizures in premenstrual syndrome and postpartum and postmenopausal dysphoria. Our data imply that ₄₃ 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 GABA_A receptors containing  subunits.

FRET-derived measurements of membrane potential provide the most robust and reliable high throughput assay of GABA_A 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 GABA_A receptors with the subunit combinations ₄₃₂ and ₄₃, this novel fluorescence technique revealed that  subunits are an important determinant of the efficacy and potency of agonists and allosteric modulators. Of particular importance was the finding that ₄₃ receptors were markedly more sensitive to inhibition by pregnanolone, suggesting that this receptor subtype could be targeted for the treatment of premenstrual syndrome.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-1279-440805; Fax: 44-1279-440390; E-mail: charles_adkins@merck.com.

Published, JBC Papers in Press, August 8, 2001, DOI 10.1074/jbc.M104318200

	ABBREVIATIONS

The abbreviations used are: GABA, -aminobutyric acid; GABA_A, -aminobutyric acid, type A; FRET, fluorescence resonance energy transfer; THIP, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol; DisBac₂(3), bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol; CC2-DMPE, chlorocoumarin-2-dimyristoyl phosphatidylethanolamine; DMCM, dimethoxy-4-ethyl--carboline-3-carboxylate; -CCE, ethyl--carboline-3-carboxylate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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