QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.070342v1 311/2/601    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Smith, A. J. Articles by Simpson, P. B. Search for Related Content PubMed PubMed Citation Articles by Smith, A. J. Articles by Simpson, P. B.

CELLULAR AND MOLECULAR

Compounds Exhibiting Selective Efficacy for Different Subunits of Human Recombinant -Aminobutyric Acid_A Receptors

Sharp and Dohme Research Laboratories, Merck, Neuroscience Research Centre, Terlings Park, Harlow, Essex, United Kingdom

Received April 20, 2004; accepted June 10, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Inhibitory GABA_A receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk^– cells stably expressing human recombinant GABA_A subunits (11–32s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl^– buffer using a voltage/ion probe reader. The influence of different subunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at 112, 122, and 132 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC₅₀ values of 2.4 ± 0.2, 2.5 ± 0.2, and 1.3 ± 0.1 µM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC₅₀ values of 8.7 ± 0.9, 9.2 ± 0.5, and 11.7 ± 1.2, and 43.7 ± 6.4, 24.8 ± 1.6, and 26.1 ± 2.4 µM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl--carboline-3-carboxylate, pentobarbital, and steroids, 5-pregnan-3-ol-20-one and 5-pregnan-3-ol-20-one, were unaffected by the isoform present in the receptor complex. However, several compounds displayed 2/3 subunit selectivity, notably loreclezole, R(–)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at 2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at 112. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 ± 14% and 109 ± 14% at 3 and 2, respectively, compared with 62 ± 6% and 56 ± 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which subunits are an important determinant of efficacy and potency.

The precise array of subunits present in the receptor complex governs the pharmacological and functional properties of receptor subtypes. The GABA agonist binding site is thought to occur at the interface of and subunits (Ebert et al., 1997), whereas affinity and efficacy at the well-characterized benzodiazepine site are influenced by residues in both and (Wingrove et al., 1997) but not (Hadingham et al., 1993) subunits. The subunit variant has been shown to influence loreclezole and etomidate sensitivity, with a single amino acid at the carboxyl-terminal end of the putative channel-lining domain TM2, being a key determinant of selectivity (Wingrove et al., 1994; Belleli et al., 1997). More recently, it has also been suggested that the subunit plays a dominant role in determining the ion selectivity of heteromeric GABA_A receptors (Jensen et al., 2002).

Several groups have described regional localization of different subunits in rat brain (Wisden et al., 1992; Miralles et al., 1999; Pirker et al., 2000). All subunits are widely distributed throughout the brain, although 2 is the most abundant, especially in cerebellum and cortex, and immuno-reactivity is also highly concentrated in thalamic nuclei with the exception of the reticular nucleus. In hippocampus, 3 is the most abundant of the subunits, but some interneurons express high levels of 2, and there is some 1 expression on both dendrites and interneurons. There are high levels of 3 expression, with little 2, in corpus striatum and in the granule cells of the olfactory bulb, whereas in the cerebellum, 2 and 3 subunits predominate over 1.

Given the wide diversity of GABA_A receptor subtypes, often in distinct neuronal circuits, and the variety of allosteric sites on the receptor through which ion channel activity can be subtly modulated, specificity is clearly key to the development of novel compounds for this therapeutic target. Progress has recently been made in dissecting functional roles of different GABA_A receptors using knockout and point mutation strategies (Rudolph et al., 2001). Evidence to date supports the notion that subunit-selective modulators may elicit desirable clinical efficacy with reduced side effects, one such example being at the benzodiazepine site where it is possible to separate anxiolysis and sedation, each being mediated via different GABA_A -subunits (McKernan et al., 2000). Identification of such compounds that can discriminate between GABA_A receptor subunits requires a functional assay with sufficiently high sensitivity to detect subtle modulation of ion channel activity.

In the present study, we have utilized a rapid ratiometric, voltage-sensitive fluorescence resonance energy transfer technique (FRET) (Gonzalez et al., 1999) to measure GABA-evoked changes in membrane potential. Using this approach, we have characterized the influence that different subunits exert on the pharmacological modulation of GABA_A receptor ion channels.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Culture. Combinations of human GABA_A receptor subunits (1x2s) were stably expressed in mouse Ltk^– fibroblast cells by transfection of the appropriate subunit cDNAs in vector pMSGneo using standard calcium phosphate transfection techniques. Cells, passaged weekly, were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA), supplemented with 10% (v/v) FetalClone II serum (Hyclone) and 1 mg/ml geneticin. For experiments, cells were removed from stock plates with 0.05% trypsin/0.53 mM EDTA solution (Invitrogen) and resuspended in DMEM supplemented with 10% FetalClone II serum but without geneticin. Cells were seeded into black-sided Porvair 96-well microtiter plates at densities of 3 to 8 x 10⁴ cells/ml in a volume of 200 µl/well and grown in the presence of 10% serum for 5 to 8 days in an incubator at 37°C. Receptor expression, which is under the control of a dexamethasone-sensitive promoter, was induced 24 h prior to experiment in the confluent cell monolayers using serum-containing DMEM supplemented with 1 µM dexamethasone (Sigma Chemical, Poole, Dorset, UK).

Fluorescence Measurement of Membrane Potential. Experiments were performed, as previously described (Adkins et al., 2001), in low-Cl^– buffer (160 mM sodium-D-gluconate, 4.5 mM potassium-D-gluconate, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM D-glucose, and 10 mM HEPES, pH 7.4). Cells were washed twice, leaving 35 µl of residual volume, and 65 µl of dyes were added to give final concentrations of 4 µM chlorocoumarin-2-dimyristoyl phosphatidylethanolamine (CC2-DMPE; FRET donor) and 1 µM bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol [DiSBAC₂(3); FRET acceptor]. Cells were dye-loaded for 30 min at room temperature in darkness, washed again, and dye solutions were then added to give a final concentration of 1 µM DiSBAC₂(3) with 0.5 mM tartrazine to quench extracellular fluorescence. Plate preparation was automated using a CCS Packard Platetrak.

Plates were then placed in a voltage/ion probe reader (Aurora Biosciences Corp., San Diego, CA), which performs automated additions using a Hamilton 2200 pipetter and records fluorescence emissions at 460 and 580 nm simultaneously from eight wells (Gonzalez et al., 1999). A 400DF15 filter was used in the excitation pathway and 460DF45 and 580DF60 filters in the respective emission pathways. Rapid ratiometric FRET measurements were made of GABA-evoked depolarizations in low-Cl^– buffer, and the ability of compounds to modulate an EC₅₀ response to GABA was examined. Experiments were performed using a two-addition protocol whereby basal fluorescence was read for 8 s before addition of test compounds, and a half-maximal concentration of GABA was added 22 s later, with fluorescence emissions recorded at 1 Hz. For assessment of the direct effect of compounds on channel opening, the initial addition was buffer only, and test compound alone was added as a second addition.

Data Analysis. Data were analyzed as previously described (Adkins et al., 2001). For each time point at each fluorescence emission wavelength, background fluorescence was subtracted (recorded from wells without cells in the same plate), and the ratio of fluorescence at 460 to 580 nm was calculated. GABA-evoked depolarizations were then expressed as a fractional change in this ratio, with the mean plateau ratio taken between 10 and 15 s after GABA addition normalized to the mean ratio over the first 6 s of recording before agonist addition. Algorithms written as Excel 97 (Microsoft, Redmond, WA) macros were used for automated calculations of fluorescence ratio, and GABA responses and concentration response curves were fitted to a four-parameter logistic equation using Prism (Prism version 2.0b; GraphPad Software Inc., San Diego, CA). Effects of compounds were expressed as percent change in the EC₅₀ GABA response in the presence of compound versus the EC₅₀ response alone, determined in control wells on the same plate.

Agonist stimulation and modulatory responses were assessed by performing one-way analysis of variance using Prism version 2.0b (GraphPad Software Inc.) followed by post hoc testing using Tukey's multiple comparison test to determine significance. Mean EC₅₀, IC₅₀, and efficacy values are presented as the arithmetic mean and S.E.M. from a number of independent and separate determinations (n), typically with at least four replicate test wells per concentration point on each plate tested.

Materials. CC2-DMPE was obtained from Aurora Biosciences Corp., DiSBAC₂(3) was from Molecular Probes (Eugene, OR), and tartrazine was obtained from Sigma. GABA, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), piperidine-4-sulfonic acid (P4S), gluconate salts, and all other GABA_A receptor modulators were also from Sigma. 2,6-diisopropylphenol (propofol) was from Aldrich, and etomidate was obtained from Janssen Pharmaceuticals (Antwerp, Belgium) as Hypnomidate, 2 mg/ml injectable solution. S(–)-Etomidate was synthesized in-house.

	Results

Top Abstract Materials and Methods Results Discussion References

 
GABA Site Ligands. Under low-chloride conditions, GABA evoked concentration-dependent depolarizations in cells loaded with CC2-DMPE and DisBac₂(3) and expressing GABA_A receptors of composition 112s, 122s, or 132s. These depolarizations were rapidly transduced into decreased FRET and, therefore, an increase in the ratio of fluorescence emission at 460 nm (f460) to that at 580 nm (f580). The fluorescence emission ratio rose to a concentration-dependent plateau within 5 s and was sustained for more than 15 s (Fig. 1). Agonist responses at each receptor subtype were of similar maximal amplitude (P > 0.05; one-way analysis of variance followed by post hoc Tukey test), although GABA exhibited slightly higher potency at 3-containing receptors (P < 0.001) (Table 1) compared with 2 and 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Concentration-dependent changes in fluorescence emission ratio with time in response to GABA at 122s receptors (data taken from a single experiment). Black bar, presence of GABA.

Other GABA site ligands also evoked concentration-dependent decreases in FRET. P4S and THIP exhibited lower potency than GABA and, in addition, P4S exhibited reduced efficacy compared with the maximum GABA response (P < 0.01; n = 4) (Table 2). THIP, however, did not exhibit significantly different efficacy compared with maximum GABA.

Influence of Subunit on Modulation of the GABA Response. The ability of compounds to modulate the response to GABA was examined using an equieffective concentration of GABA (EC₅₀), predetermined for each receptor subtype. As previously reported (Hadingham et al., 1993), benzodiazepine site ligands did not discriminate between receptor isoforms containing different subunits. At the maximal concentration tested (10 µM), the inverse agonist methyl 6,7-dimethoxy-4-ethyl--carboline-3-carboxylate negatively modulated GABA responses at 112, 122, and 132 receptors, with maximal efficacies of –48 ± 4% (n = 5), –42 ± 6% (n = 5), and –42 ± 6% (n = 3), respectively (Fig. 2). In contrast, the anticonvulsant loreclezole exhibited subtype selectivity, with quantitatively greater maximal efficacy and potency at 2- and 3-versus 1-containing receptors (Fig. 2). There was little potentiation of 112 receptors up to 10 µM (15 ± 4%, n = 7), whereas 122 and 132 receptor responses were potentiated by 56 ± 3% (n = 7) and 62 ± 6% (n = 4), respectively, with EC₅₀ values of 1.9 and 0.6 µM. Neither compound elicited any direct activation of the receptor in the absence of GABA.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Cells were pretreated with different concentrations of the benzodiazepine site inverse agonist methyl 6,7-dimethoxy-4-ethyl--carboline-3-carboxylate () or the anticonvulsant loreclezole () before addition of GABA at a half-maximal concentration (EC50), previously established for each receptor subtype. Results (n = 3–7 determinations) are shown as mean percent modulation of the control EC50 response ± S.E.M. Loreclezole selectively potentiated 2- and 3-containing receptors. Neither compound elicited any direct effect on the ion channel alone (data not shown).

Modulation and Activation of _1x2s GABA_A Receptors by Anesthetics, Steroids, and Anti-Inflammatory Agents. The i.v. anesthetic propofol (Fig. 3A) and the non-volatile anesthetic barbiturate pentobarbital (Fig. 3B) potentiated the EC₅₀ GABA response at all receptor subtypes. The maximal efficacies and potencies for propofol were not significantly different for 1-, 2-, and 3-containing receptors, being 123 ± 21, 132 ± 29, and 128 ± 24% and 3.8 ± 1.2, 2.4 ± 0.6, and 1.6 ± 0.1 µM, respectively (P > 0.05; n = 5). Pentobarbital was approximately an order of magnitude less potent than propofol, with EC₅₀ values of 22.6 ± 2.3, 22.0 ± 1.9, and 13.4 ± 2.5 µM, and maximal efficacies of 133 ± 29, 168 ± 26, and 140 ± 41%, respectively (n = 3). Both propofol and pentobarbital directly activated the ion channel in the absence of GABA at concentrations approximately 10-fold higher than those eliciting potentiation of the GABA response (EC₅₀ values of 27.2 ± 2.0, 25.3 ± 1.7, and 20.3 ± 3.0 µM, n = 6–9; and 119 ± 7.5, 117 ± 15.9, and 75 ± 3.0 µM, n = 3–6, respectively). For all compounds where a significant direct effect of the compound on the ion channel in the absence of GABA was observed, the modulation effects shown represent the total effect of the compound, given the absence of tools to block this direct component. Thus, for pentobarbital, at concentrations above 100 µM, a significant portion of the modulatory effect is likely to be due to a direct effect on the channel itself.

View larger version (23K): [in this window] [in a new window]   Fig. 3. The potencies and efficacies for potentiation of the GABA EC50 response and direct channel gating by the i.v. anesthetic propofol (A) and the barbiturate pentobarbital (B) were not markedly affected by the isoform present in the receptor complex. Propofol (n = 5 determinations) was an order of magnitude more potent than pentobarbital (n = 3 determinations) in potentiating GABA. Both compounds elicited direct channel activation at all subtypes at concentrations approximately 10-fold higher than those that potentiated GABA (n = 3 determinations each). The modulation graph represents the total effect of the compounds.

The anesthetic agent etomidate similarly both potentiated the EC₅₀ GABA response and, at higher concentrations, caused direct activation (Fig. 4A). Like loreclezole, etomidate also displayed functional selectivity for 2/3 subunits over 1, having both increased potency (EC₅₀ values 0.5 ± 0.1 and 0.3 ± 0.1 µM, respectively) and higher maximal efficacy (152 ± 20 and 158 ± 23% potentiation, respectively) than at 112 receptors (EC₅₀ 2.9 ± 0.8 µM; efficacy 107 ± 19%; n = 4). Etomidate evoked a higher degree of potentiation of the GABA response than loreclezole at all three subunit combinations examined. Unlike loreclezole, etomidate elicited direct effects on GABA_A receptors, and this direct activation of the ion channel at higher concentrations also displayed subunit selectivity (n = 6). Etomidate exhibited enantiomeric selectivity, with the S(+)-enantiomer displaying reduced potency and no subunit selectivity (Fig. 4B), in contrast to the R(–)-enantiomer (Fig. 4A). The S(+)-enantiomer could not be tested at high enough concentrations to determine whether there was any direct activation.

View larger version (18K): [in this window] [in a new window]   Fig. 4. The anesthetic agent R(–)-etomidate exhibited higher potency and efficacy for potentiation of GABA responses at receptors containing a 2 or a 3 subunit (mean ± S.E.M. from four independent experiments) and also elicited direct ion channel activation at higher concentrations (n = 6), potency and efficacy being reduced at 1-containing receptors (A); etomidate also exhibited stereoselectivity with the S(+)-enantiomer displaying lower potency and no subunit selectivity (B).

The steroid pharmacology of these GABA_A receptor isoforms was also investigated. The progesterone metabolite 5-pregnan-3-ol-20-one, together with 5-pregnan-3-ol-20-one and 5-pregnan-3-ol-20-one, were all tested for their ability to modulate ion channel opening (Fig. 5). 5-Pregnan-3-ol-20-one robustly potentiated GABA responses and did not discriminate between different subunits, eliciting quantitatively similar maximal efficacies at all receptors with little difference in potency (Fig. 5a). In contrast, 5-pregnan-3-ol-20-one had no significant effect on the GABA response except at the top concentration (200 µM) where there was a modest inhibition (1, –18 ± 4%; 2, –38 ± 7%; 3, –35 ± 8%; n = 9) (Fig. 5b). 5-Pregnan-3-ol-20-one elicited potentiation of all three receptor subtypes with similar potency and maximal efficacy (Fig. 5c).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Modulation of GABA EC50 responses at 1x2s GABAA receptors in response to steroids. a, response to 5-pregnan-3-ol-20-one (n = 3 determinations. b, 5-pregnan-3-ol-20-one (n = 3 determinations). c, 5-pregnan-3-ol-20-one (n = 4 determinations).

A group of nonsteroidal anti-inflammatory agents were also examined for modulatory and direct effects on GABA_A receptors (chemical structures shown in Fig. 6). The aminoarylcarboxylic acid derivatives mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, and niflumic acid and the salicylic acid derivatives diflunisal and olsalazine all showed activity at GABA_A receptors. Except for olsalazine, the anti-inflammatories all selectively potentiated 122s and 132s receptors, exhibiting varying levels of efficacy at 2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at 112. The ability of compounds to modulate the EC₅₀ response to GABA is shown in Table 3. Diflunisal was the most efficacious of these compounds, eliciting greater potentiation than loreclezole (90 ± 14 and 109 ± 14% at 3 and 2, respectively, compared with 62 ± 6 and 56 ± 3%), although it was an order of magnitude less potent. Niflumic acid exhibited the lowest efficacy. The aminoarylcarboxylic acid derivatives were more potent modulators than the salicylic acid derivatives. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity and elicited some direct activation at the top concentration tested (200 µM). There was no direct receptor activation observed up to 200 µM with niflumic acid or flufenamic acid, a very modest direct effect at the top concentration with mefenamic acid and diflunisal, whereas a more robust direct effect was observed for meclofenamic (Fig. 7) and tolfenamic (Fig. 8) acids.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Structures of loreclezole, etomidate, and anti-inflammatory agents evaluated as GABAA receptor modulators.

View larger version (10K): [in this window] [in a new window]   Fig. 7. The nonsteroidal anti-inflammatory agent meclofenamic acid selectively potentiated 122s and 132s receptors with similar potency and similar maximal efficacy, while displaying inverse agonism at 1 (n = 7 determinations). There was some direct activation with this compound at concentrations approximately 10-fold higher than those at which potentiation was observed (n = 4 determinations).

View larger version (10K): [in this window] [in a new window]   Fig. 8. A related anti-inflammatory agent, tolfenamic acid, also selectively potentiated GABA responses at 122s and 132s receptors in a concentration-dependent manner (n = 9 determinations) and elicited direct activation at the top concentration tested (200 µM) (n = 4 determinations).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we have performed the most comprehensive study yet undertaken of the effect of subunit expression on the activation and modulation of GABA_A receptor subtypes. We demonstrate the importance of subunits in influencing the action of a variety of agents, highlighted by the marked differences in efficacy and potency of loreclezole, etomidate, and multiple nonsteroidal anti-inflammatory agents, the GABA_A receptor selectivity of some of which are reported here for the first time. An earlier study examining GABA-evoked currents recorded in oocytes expressing rat cortex mRNA (Woodward et al., 1994) was the first to suggest that some anti-inflammatory agents had the potential to modulate GABA_A receptors, although the precise receptor subunit composition was unclear, and there was no information about receptor selectivity. Many of the compounds displaying subunit selectivity are used clinically, and it will be interesting to explore further whether differences in their efficacy profiles underlie some of their distinct physiological effects.

Evidence suggests that nonsteroidal anti-inflammatories can effectively cross the blood-brain barrier (Bannwarth et al., 1989). This, together with the relatively high therapeutic plasma concentrations that are achieved in the region of 80 µM and 20 to 70 µM for mefenamic and niflumic acids, respectively (Halliwell et al., 1999; Sinkkonen et al., 2003), suggests that appreciable brain levels might be achieved. Indeed, observations of antiepileptogenic effects (Wallenstein, 1991) and adverse events associated with anti-inflammatory overdose are consistent with activity at central GABA_A receptors. Given that low micromolar concentrations are sufficient to potentiate 2/3-containing GABA_A receptors, this suggests that modulatory effects could occur at clinically relevant concentrations. In addition, the anti-inflammatory-sensitive 122 receptor subtype is the largest GABA_A receptor population in mammalian brain (McKernan and Whiting, 1996). As well as activity at GABA_A receptor ion channels, fenamate nonsteroidal anti-inflammatories have also been reported to have activity at a variety of other ion channels (e.g., Greenwood and Large, 1995; Malykhina et al., 2002).

Application of FRET techniques to the study of GABA_A receptors has been previously described (Adkins et al., 2001). Clearly, in terms of agonist time course, these FRET responses have a slower onset than those determined by more direct electrophysiological measures of channel opening, with peak currents attained within 5 s versus millisecond responses using patch-clamp techniques. Although the GABA response is likely to occur rapidly, changes in FRET are downstream of this and, thus, are a slower readout of channel function. The current technique is influenced by the speed of voltage-dependent redistribution DiSBAC₂(3) in the membrane, which occurs subsequent to the flow of chloride ions and membrane depolarization. However, potency values obtained using voltage/ion probe reader fluorescence measurements for GABA-evoked depolarizations and allosteric modulation at a variety of GABA_A receptors agree well with values reported from other assays including electrophysiology (e.g., Wafford et al., 1994; Smith et al., 2001). In addition, this technique offers the potential for much increased throughput, allowing scope to more widely explore the pharmacology of different recombinant receptors.

Recent studies of murine 3 homomeric GABA_A ion channels indicate these GABA-insensitive receptors can still respond to pentobarbital, propofol, etomidate, and pregnanolone (Wooltorton et al., 1997), although the properties of murine 1 homomers are somewhat different. This appears to be due to a single amino acid, asparagine-265, at the extracellular end of M2 (Cestari et al., 2000), the same residue that regulates potentiation by etomidate (Belleli et al., 1997) but not pentobarbital in heteromeric receptors. This amino acid has also been implicated in the action of loreclezole (Wingrove et al., 1994) and mediates potentiation by high concentrations of -carbolines (Stevenson et al., 1995). Although the presence of a 2 or 3 subunit is clearly a prerequisite for loreclezole efficacy, different or subunits reportedly influence the level of potentiation (Wafford et al., 1994). In a detailed study of one particular anti-inflammatory agent at different recombinant receptors (Halliwell et al., 1999), the same residue was also implicated in subunit-selective modulation of GABA_A receptors by mefenamic acid. Additionally, Sinkkonen et al. (2003) reported that potentiation of 122 receptors by niflumic acid depended on the presence of a 2 subunit, also found to influence mefenamic acid modulation (Halliwell et al., 1999). Antagonism of the 622 receptor subtype by niflumic acid has also been reported (Sinkkonen et al., 2003), and substitution of an 4 subunit reduced mefenamic potentiation of 122 receptors by 50% (Whittemore et al., 1996).

Pentobarbital, propofol, and etomidate all elicited quantitatively larger potentiations of the GABA response than loreclezole, anti-inflammatories, and steroids. Although the nature of the subunit is clearly critical for both potentiation and GABA-mimetic activity of etomidate, with lower potency and efficacy evident for 1-containing receptors, the actions of propofol, pentobarbital, and steroids were found in our study to be similar irrespective of the isoform present in the receptor complex. However, evidence for direct involvement of subunit residues in the action of propofol comes from a study showing that methionine 286 in transmembrane domain 3 of the subunit controlled a binding cavity for propofol (Krasowski et al., 2001) and was implicated in potentiation of GABA responses but not in direct activation. This may imply the two phenomena are mediated via separate sites, a view supported by Siegwart et al. (2002), who observed differential structural requirements for modulatory and direct effects of a variety of general anesthetics. There is also evidence that a glycine residue at the entrance to TM1 of the 2 subunit is involved in channel gating and anesthetic modulation (Carlson et al., 2000). In the current study, pentobarbital, etomidate, and propofol all had pronounced effects on direct activation at approximately 10-fold higher concentrations than those which potentiated the GABA response.

With regard to pentobarbital, the affinity of potentiation electrophysiologically in oocytes was little affected by altering or subunits, although the maximal degree of potentiation was influenced by the nature of the former but not the latter (Thompson et al., 1996), in agreement with the present results. With regard to direct activation by pentobarbital, the subunit influenced both the affinity and efficacy of the direct effect, whereas the nature of the subunit exerted a lesser influence on these parameters, only showing differences when coexpressed with 1 but not with 6. This is in contrast to the current observations and may reflect cell-type-specific differences. Although the subunit appears to have little impact on the action of steroids, it clearly plays a role in mediating their effects since modulation is preserved even at homomeric subunit-containing receptors (Wooltorton et al., 1997).

Many compounds examined in the present study showed evidence of declining responses at high concentrations (e.g., Figs. 3, a and b, 4a, 7, and 8), and this was evident both for modulatory and direct effects. Similar observations have been made in electrophysiological studies of the action of pentobarbital, etomidate, and mefenamic acid (Thompson et al., 1996; Belleli et al., 1997; Halliwell et al., 1999). Previous studies have identified several different effects with respect to the action of anesthetic agents: a potentiation of the GABA response, direct activation of the ion channel, and, at higher concentrations, a block of the GABA chloride channel. It is likely, at higher concentrations of test agents, that multiple processes may be occurring simultaneously that may limit the amount of potentiation that can be observed.

Etomidate showed stereoselectivity in potentiating GABA responses, with the S(–)-enantiomer exhibiting lower potency and no subunit selectivity, unlike the R(+)-enantiomer. Stereoselectivity has also been reported for steroids with multiple chiral centers (Covey et al., 2000). The latter study indicates that 5-reduced steroids show a much higher degree of enantiospecificity in their actions as modulators and as anesthetics than 5-reduced steroids. The limited effect here of 5-pregnan-3-ol-20-one agrees with previous published data describing the stereoselectivity of the 3-hydroxy group, which must be in the configuration for potent receptor modulation (El-Etr et al., 1998; Hawkinson et al., 1998).

An additional compound which is a derivative of salicylic acid, salicylidene salicylhydrazide, has recently been reported by us to also display subunit selectivity (Thompson et al., 2004), being a selective inhibitor of 1-containing receptors. This further supports the finding that subunits are an important determinant of efficacy and potency for a variety of allosteric modulators and direct activators of human GABA_A receptor ion channels. In the current study, we have used a novel fluorescence technique that provides rapid and sensitive measurements of GABA_A receptor function to examine the influence of differing subunit expression on the potency and efficacy of a variety of ligands. Several compounds discriminated 2/3 from 1-containing receptors including the anticonvulsant loreclezole, the anesthetic etomidate, and a relatively diverse group of anti-inflammatory agents.

	Acknowledgements

 
We would like to extend our thanks to Matthew Leveridge and Julie Kerby, who provided cell culture support for this work.

	Footnotes

 
doi:10.1124/jpet.104.070342.

ABBREVIATIONS: FRET, fluorescence resonance energy transfer; DMEM, Dulbecco's modified Eagle's medium; CC2-DMPE, chlorocoumarin-2-dimyristoyl phosphatidylethanolamine; DiSBAC₂(3), bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; P4S, piperidine-4-sulfonic acid.

Address correspondence to: Alison J. Smith, Sharp and Dohme Research Laboratories, Merck, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK. E-mail: alison_smith{at}merck.com.

	References

Top Abstract Materials and Methods Results Discussion References

 

Adkins CE, Pillai GV, Kerby J, Bonnert TP, Haldon C, McKernan RM, Gonzalez JE, Oades K, Whiting PJ, and Simpson PB (2001) 43 GABA_A receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential. J Biol Chem 276: 38934–38939.[Abstract/Free Full Text]

Bannwarth B, Netter P, Pourel J, Royer RJ, and Gaucher A (1989) Clinical pharmacokinetics of nonsteroidal anti-inflammatory drugs in the cerebrospinal fluid. Biomed Pharmacother 43: 121–126.[CrossRef][Medline]

Belleli D, Lambert JL, Peters JA, Wafford K, and Whiting PJ (1997) The interaction of the general anesthetic etomidate with the -aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci USA 94: 11031–11036.[Abstract/Free Full Text]

Bollan K, King D, Robertson LA, Brown K, Taylor PM, Moss SJ, and Connolly CN (2003) GABA_A receptor composition is determined by distinct assembly signals within alpha and beta subunits. J Biol Chem 278: 4747–4755.[Abstract/Free Full Text]

Carlson BX, Engblom AC, Kristiansen U, Schousboe A, and Olsen RW (2000) A single glycine residue at the entrance to the first membrane-spanning domain of the -aminobutyric acid type A receptor 2 subunit affects allosteric sensitivity to GABA and anesthetics. Mol Pharmacol 57: 474–484.[Abstract/Free Full Text]

Cestari IN, Min KT, Kulli JC, and Yang J (2000) Identification of an amino acid defining the distinct properties of murine 1 and 3 subunit-containing GABA_A receptors. J Neurochem 74: 827–838.[CrossRef][Medline]

Covey DF, Nathan D, Kalkbrenner M, Nilsson KR, Hu Y, Zorumski CF, and Evers AS (2000) Enantioselectivity of pregnanolone-induced -aminobutyric acid_A receptor modulation and anaesthesia. J Pharmacol Exp Ther 293: 1009–1116.[Abstract/Free Full Text]

Ebert B, Thompson SA, Saounatsou K, McKernan R, Krogsgaard-Larsen P, and Wafford K (1997) Differences in agonist/antagonist binding affinity and receptor transduction using recombinant human -aminobutyric acid type A receptors. cells. Mol Pharmacol 52: 1150–1156.[Abstract/Free Full Text]

El-Etr M, Akwa Y, Robel P, and Baulieu EE (1998) Opposing effects of different steroid sulfates on GABA_A receptor-mediated chloride uptake. Brain Res 790: 334–338.[CrossRef][Medline]

Farrar SJ, Whiting PJ, Bonnert TP, and McKernan RM (1999) Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. J Biol Chem 274: 10100–10104.[Abstract/Free Full Text]

Fritschy J-M and Mohler H (1995) GABA_A receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359: 154–194.[CrossRef][Medline]

Gonzalez JE, Oades K, Leychkis Y, Harootunian A, and Negulescu PA (1999) Cell-based assays and instrumentation for screening ion channel targets. Drug Discov Today 4: 431–439.[CrossRef][Medline]

Greenwood IA and Large WA (1995) Comparison of the effects of fenamates on Ca-activated chloride and potassium currents in rabbit portal vein smooth muscle cells. Br J Pharmacol 116: 2939–2948.[Medline]

Hadingham KL, Wingrove PB, Wafford KA, Bain C, Kemp JA, Palmer KJ, Wilson AW, Wilcox AS, Sikela JM, Ragan CI, et al. (1993) Role of the subunit in determining the pharmacology of human -aminobutyric acid type A receptors. Mol Pharmacol 44: 1211–1218.[Abstract]

Halliwell RF, Thomas P, Patten D, James CH, Martinez-Torres A, Miledi R, and Smart TG (1999) Subunit-selective modulation of GABA_A receptors by the non-steroidal anti-inflammatory agent, mefenamic acid. Eur J Neurosci 11: 2897–2905.[CrossRef][Medline]

Hawkinson JE, Acosta-Burruel M, Yang KC, Hogenkamp DJ, Chen J-S, Lan NC, Drewe JA, Whittemore ER, Woodward RM, Carter RB, et al. (1998) Substituted 3-phenylethynyl derivatives of 3-hydroxy-5-pregnan-20-one: remarkably potent neuroactive steroid modulators of -aminobutyric acid type A receptors. J Pharmacol Exp Ther 287: 198–207.[Abstract/Free Full Text]

Jensen ML, Timmermann DB, Johansen TH, Schousboe A, Varming T, and Ahring PK (2002) The subunit determines the ion selectivity of the GABA_A receptor. J Biol Chem 277: 41438–41447.[Abstract/Free Full Text]

Krasowski MD, Nishikawa K, Nikolaeva N, Lin A, and Harrison NL (2001) Methionine 286 in transmembrane domain 3 of the GABA_A receptor subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. Neuropharmacology 41: 952–964.[CrossRef][Medline]

Malykhina AP, Shoeb F, and Akbarali HI (2002) Fenamate-induced enhancement of heterologously expressed HERG currents in Xenopus oocytes. Eur J Pharmacol 452: 269–277.[CrossRef][Medline]

McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S, Myers J, Cook G, Ferris P, et al. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA_A receptor 1 subtype. Nat Neurosci 3: 587–592.[CrossRef][Medline]

McKernan RM and Whiting PJ (1996) Which GABA_A receptor subtypes really occur in the brain? Trends Neurol Sci 19: 139–143.[CrossRef][Medline]

Mehta AK and Ticku MK (1999) An update on GABA_A receptors. Brain Res Rev 29: 196–217.[CrossRef][Medline]

Miralles CP, Li M, Mehta AK, Khan ZU, and De Blas AL (1999) Immunocytochemical localization of the 3 subunit of the -aminobutyric acid_A receptor in the rat brain. J Comp Neurol 413: 535–548.[CrossRef][Medline]

Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, and Sperk G (2000) GABA_A receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101: 815–850.[CrossRef][Medline]

Rudolph U, Crestani F, and Mohler H (2001) GABA_A receptor subtypes: dissecting their pharmacological functions. Trends Pharmacol Sci 22: 188–194.[CrossRef][Medline]

Siegwart R, Jurd R, and Rudolph U (2002) Molecular determinants for the action of general anesthetics at recombinant -aminobutyric acid_A receptors. J Neurochem 80: 140–148.[CrossRef][Medline]

Sinkkonen ST, Mansikkamaki S, Moykkynen T, Luddens H, Uusi-Oukari M, and Korpi ER (2003) Receptor subtype-dependent positive and negative modulation of GABA_A receptor function by niflumic acid, a nonsteroidal anti-inflammatory drug. Mol Pharmacol 64: 753–763.[Abstract/Free Full Text]

Smith AJ, Alder L, Silk J, Adkins C, Fletcher AE, Scales T, Kerby J, Marshall G, Wafford KA, McKernan RM, et al. (2001) Effect of alpha subunit on allosteric modulation of ion channel function in stably expressed human recombinant -aminobutyric acid_A receptors determined using ³⁶Cl ion flux. Mol Pharmacol 59: 1108–1118.[Abstract/Free Full Text]

Stevenson A, Wingrove PB, Whiting PJ, and Wafford KA (1995) -Carboline -aminobutyric acid A receptor inverse agonists modulate -aminobutyric acid via the loreclezole binding site as well as the benzodiazepine site. Mol Pharmacol 48: 965–969.[Abstract]

Thompson SA, Wheat L, Brown NA, Wingrove PB, Pillai GV, Whiting PJ, Adkins C, Woodward CH, Smith AJ, Simpson PB, et al. (2004) Salicylidene salicylhydrazide, a selective inhibitor of 1 containing GABA_A receptors. Br J Pharmacol 142: 97–106.[CrossRef][Medline]

Thompson SA, Whiting PJ, and Wafford KA (1996) Barbiturate interactions at the human GABA_A receptor: dependence on receptor subunit combination. Br J Pharmacol 117: 521–527.[Medline]

Wafford KA, Bain CJ, Quirk K, McKernan RM, Wingrove PB, Whiting PJ, and Kemp JA (1994) A novel allosteric modulatory site on the GABA_A receptor subunit. Neuron 12: 775–782.[CrossRef][Medline]

Wallenstein MC (1991) Attenuation of epileptogenesis by non-steroidal anti-inflammatory drugs in the rat. Neuropharmacology 30: 657–663.[CrossRef][Medline]

Whiting PJ, Bonnert TP, McKernan RM, Farrar S, Bourdelles BL, Heavens RP, Smith DW, Hewson L, Rigby MR, Sirinathsinghji DJ, et al. (1999) Molecular and functional diversity of the expanding GABA_A receptor gene family. Ann NY Acad Sci 868: 645–653.[CrossRef][Medline]

Whittemore ER, Yang W, Drewe JA, and Woodward RM (1996) Pharmacology of the human -aminobutyric acid A receptor 4 subunit expressed in Xenopus laevis oocytes. Mol Pharmacol 50: 1364–1375.[Abstract]

Wingrove PB, Thompson SA, Wafford KA, and Whiting PJ (1997) Identification of key amino acids in the gamma subunit of the GABA_A receptor which contribute to the benzodiazepine binding site. Mol Pharmacol 52: 874–881.[Abstract/Free Full Text]

Wingrove PB, Wafford KA, Bain C, and Whiting PJ (1994) The modulatory action of loreclezole at the -aminobutyric acid type A receptor is determined by a single amino acid in the 2 and 3 subunit. Proc Natl Acad Sci USA 91: 4569–4573.[Abstract/Free Full Text]

Wisden W, Laurie DJ, Monyer H, and Seeburg PH (1992) The distribution of 13 GABA_A receptor subunit mRNAs in the rat brain: I. Telencephalon, diencephalon, mesencephalon. J Neurosci 12: 1040–1062.[Abstract]

Woodward RM, Polenzani L, and Miledi R (1994) Effects of fenamates and other nonsteroidal anti-inflammatory drugs on rat brain GABA_A receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 268: 806–817.[Abstract/Free Full Text]

Wooltorton JRA, Moss SJ, and Smart TG (1997) Pharmacological and physiological characterization of murine homomeric 3 GABA_A receptors. Eur J Neurosci 9: 2225–2235.[CrossRef][Medline]

This article has been cited by other articles:

				R. W. Olsen and W. Sieghart International Union of Pharmacology. LXX. Subtypes of {gamma}-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update Pharmacol. Rev., September 1, 2008; 60(3): 243 - 260. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.070342v1 311/2/601    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Smith, A. J. Articles by Simpson, P. B. Search for Related Content PubMed PubMed Citation Articles by Smith, A. J. Articles by Simpson, P. B.