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

Inhibitory GABAA receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk cells stably expressing human recombinant GABAA subunits ( 1 1–3 2s) 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 lowCl 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 1 1 2, 1 2 2, and 1 3 2 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 M, 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 M, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethylcarboline-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 antiinflammatory 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 1 1 2. 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. GABA is the predominant inhibitory neurotransmitter in mammalian central nervous system, and ligand-gated GABAA receptor ion channels are the target for a wide range of therapeutic agents including benzodiazepines, barbiturates, steroids, convulsants, and anesthetics (reviewed in Mehta and Ticku, 1999). GABAA receptors are pentameric assemblies of multiple subunits ( 1-6, 1-3, 1-3, , , , and ; Whiting et al., 1999) with an integral chloride channel and a variety of allosteric binding sites through which rapid inhibitory synaptic neurotransmission can be modulated. Subunit assembly and stoichiometry studies have revealed a variety of receptor subunit combinations that are differentially expressed in mammalian brain (Fritschy and Mohler, 1995; McKernan and Whiting, 1996), with the most abundant receptor subtypes generally having a 2 :2 :1 stoichiometry (Farrar et al., 1999). Multiple subunits provide the potential for enormous structural diversity, although evidence suggests differential assembly signals within and subunits operate to restrict the number of possible combinations (Bollan et al., 2002). The largest GABAA receptor population in rat brain has an 1 2 2 composition, whereas 2 3 2 and 3 2/ 3 together constitute the next most prevalent subtypes (McKernan and Whiting, 1996). 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 Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.070342. ABBREVIATIONS: FRET, fluorescence resonance energy transfer; DMEM, Dulbecco’s modified Eagle’s medium; CC2-DMPE, chlorocoumarin2-dimyristoyl phosphatidylethanolamine; DiSBAC2(3), bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol; P4S, piperidine-4-sulfonic acid. 0022-3565/04/3112-601–609$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 311, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 70342/1172609 JPET 311:601–609, 2004 Printed in U.S.A. 601 at A PE T Jornals on M ay 5, 2017 jpet.asjournals.org D ow nladed from 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 GABAA 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 immunoreactivity 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 GABAA 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 GABAA 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 GABAA -subunits (McKernan et al., 2000). Identification of such compounds that can discriminate between GABAA 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 GABAevoked changes in membrane potential. Using this approach, we have characterized the influence that different subunits exert on the pharmacological modulation of GABAA receptor ion channels. Materials and Methods Cell Culture. Combinations of human GABAA receptor subunits ( 1 x 2s) 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 10 cells/ml in a volume of 200 l/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 M dexamethasone (Sigma Chemical,