Introduction

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GABA_A receptors mediate most of the inhibitory synaptic transmission in the central nervous system and are the principal target of neuroactive drugs used in the treatment of anxiety, insomnia, and epilepsy. Based on their genetic and structural relatedness, GABA_A receptors belong to the nicotinic superfamily of neurotransmitter receptor ion channels, which also includes nicotinic acetylcholine (nACh), serotonin type-3 (5-HT₃), and glycine receptors. GABA_A receptors are GABA-gated Cl ion channels typically composed of two  (1-6), two  (1-3), and one  (1-3) or 1 subunit in a pentameric assembly (Sieghart, 1995; Barnard et al., 1998; Mehta and Ticku, 1999). Among the many drugs that act at these receptors are the benzodiazepines, allosteric modulators that enhance GABA_A receptor signaling by binding to a site at the - subunit interface (Gunther et al., 1995; Sigel and Buhr, 1997). Transgenic studies indicate that benzodiazepines exert their sedative effects and partly their anticonvulsant effects through 122 receptors (Rudolph et al., 1999; McKernan et al., 2000) and their anxiolytic effects through 222 receptors (Low et al., 2000). Thus, it may be possible for a drug to elicit only a desired action by targeting a particular receptor subtype.

Zopiclone is a cyclopyrrolone, a class of nonbenzodiazepine drugs that have high affinity for the benzodiazepine binding site but, relative to benzodiazepines, produce comparable anxiolytic effects with less sedation, muscle relaxation, or addictive potential (Piot et al., 1990; Sanger et al., 1994; Karle and Nielsen, 1998). Racemic zopiclone, which consists of (R)- and (S)-enantiomers, acts at the benzodiazepine site to enhance GABA_A receptor binding and function (Im et al., 1993; Davies et al., 2000). Although the selectivity of zopiclone is not fully characterized, it is known to act at 2-bearing GABA_A receptors, including 122 (Im et al., 1993; Reynolds and Maitra, 1996; Davies et al., 2000). Recent behavioral studies in rodents suggest that the sedative and anxiolytic activities of (R,S)-zopiclone are produced mainly by the (S)-enantiomer (Carlson et al., 2001), which is metabolized in vivo to (S)-desmethylzopiclone (SEP-174559). SEP-174559 is active on its own and was found selectively to produce anxiolytic and anticonvulsant effects; doses 100 times higher were required to impair locomotor activity or motor coordination (Carlson et al., 2001). Yet, the activity of SEP-174559 and its selectivity for various subtypes of the GABA_A receptor have not been described.

The present study profiles the benzodiazepine-like actions of SEP-174559 in comparison with zopiclone at various GABA_A receptor subtypes and further defines the spectrum of effects of benzodiazepine-site ligands at other neurotransmitter receptor ion channels in both the nicotinic and glutamate receptor superfamilies.

	Experimental Procedures

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Cell Cultures and Transfections. Human embryonic kidney 293 fibroblasts (HEK293, CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Carlsbad, CA) and incubated at 37°C in a 5% CO₂ environment. Cells were plated into 25-cm² flasks (Falcon Plastics, Oxnard, CA) and passaged twice weekly to fresh flasks. Excess cells were removed, plated onto poly-d-lysine-coated 35-mm dishes (Nalge Nunc, Naperville, IL), and cotransfected the following day with cDNA plasmids encoding receptor subunits and enhanced green fluorescent protein at a 9:1 ratio. Transfections were done using the LipofectAMINE PLUS reagents (Invitrogen). Patch-clamp recordings were obtained 12 to 48 h post-transfection. Studies used mammalian cDNA expression vectors encoding rat GABA_A receptor 1 (accession no. L08490), 2 (L08491), 3 (L08492), 2 (X15467), 1 (X57514), and 2 (L08497) subunits, rat nACh receptor 3 (L31621) and 4 (U42976) subunits, the mouse 5-HT_3A receptor subunit (M74425), rat NMDA receptor NR1 (X63255) and NR2B (M91562) subunits, rat GluR1_flip AMPA receptor subunit (M38060), and rat GluR6 kainate receptor subunit (Z11715).

Patch-Clamp Recording. Cells were continuously superfused with standard extracellular solution containing 150 mM NaCl, 3 mM KCl, 5 mM HEPES, 1 mM MgCl₂, 1.8 mM CaCl₂, 10 mM glucose, and 0.1 mg ml¹ phenol red, pH 7.3. Recording microelectrodes were fabricated from thin-walled borosilicate glass capillary tubes (TW150F; World Precision Instruments, New Haven, CT) having resistances of 2 to 4 M when filled with an internal solution containing 135 mM CsCl, 10 mM CsF, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl₂, and 0.5 mM CaCl₂, pH 7.2. Whole-cell and outside-out patch recordings were performed in voltage clamp at a holding potential of 70 mV using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Current signals were filtered at 1 to 3 kHz with an eight-pole Bessel filter (Cygnus Technology, Delaware Water Gap, PA), digitized at 1 to 20 kHz, and stored on a Macintosh PowerPC-G3 computer using an ITC-16 interface (InstruTECH Corporation, Port Washington, NY) under the control of the data acquisition and analysis program Synapse (Synergistic Research Systems, Silver Spring, MD).

Rapid Solution Exchange. Rapid drug applications were achieved in two ways. In whole-cell recordings of most receptors, a multivalve solution exchange system was used. Solutions were driven by a syringe pump at a rate of 2 ml min¹ through a flowpipe having either four or eight inputs glued together within ~1 mm of a common output. Solution flow-through in each channel was controlled by a set of upstream three-way solenoid valves (Lee Co., Westbrook, CT). Switching between control and drug solutions was achieved by opening and closing the appropriate valves under computer control. The rate of solution exchange using this system was 5 ms as determined from open-tip junction currents but was further limited by cell diameter. In outside-out patch recordings with GluR1 and GluR6 receptors, the ultrafast solution exchange needed to resolve the fast response, and kinetic properties of these receptors was achieved using an LSS-3100 piezotranslator (Burleigh, Fishers, NY). Control and agonist solutions were driven simultaneously at a rate of 0.3 ml min¹ through the two parallel barrels of a theta tube. The membrane patch was positioned in the control stream near the solution interface and a piezotranslator was used to rapidly move the theta tube ~50 µm such that the solution interface moved across the patch. Inputs were then switched to solutions containing drug and retested. The rate of solution exchange in this system was 100 µs as determined from open-tip junction currents measured at the end of each experiment.

Data Analysis. Agonist dose-response data were normalized to saturation values for each cell and fit by the logistic equation I = I_max/(1 + (EC₅₀/[agonist])^n_H), where I_max is the maximal current at saturating agonist concentrations, EC₅₀ is the concentration of agonist that elicits a half-maximal response, and n_H is the Hill coefficient or steepness factor; these parameters were derived using an iterative least-squares fitting algorithm.

Materials. Reagents were from Sigma-Aldrich (St. Louis, MO) or Sigma/RBI (Natick, MA). SEP-174559, (R,S)-zopiclone, and the zopiclone enantiomers were kindly provided by Sepracor (Marlborough, MA). Agonist and drug stocks were prepared in 0.05 M HEPES buffer, pH 7.4, except (R,S)-zopiclone, (S)-zopiclone, (R)-zopiclone, alprazolam, and diazepam were prepared at 20 mM in dimethyl sulfoxide; the final concentration of dimethyl sulfoxide was 0.1%, which was tested and found by itself to have no activity on the receptors examined in this study. Agonist solutions for dose-response analyses were prepared freshly by serial dilution.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Racemic zopiclone [(R,S)-zopiclone] has been shown to enhance GABA_A receptor currents via the benzodiazepine site (Im et al., 1993; Davies et al., 2000). The molecular actions of its principal metabolite, SEP-174559, have not been described. To profile the activity and GABA_A receptor selectivity of SEP-174559, GABA-evoked currents were tested by whole-cell patch-clamp recording in transfected HEK293 cells expressing various recombinant GABA_A receptor subtypes. Effects on related and unrelated neurotransmitter receptor ion channels were also tested to assess the specificity of drug action. In many cases, the actions of (R,S)-zopiclone or other benzodiazepine-site ligands were also tested for comparison.

GABA_A Receptor Actions. To test the influence of the GABA_A receptor  subunit, HEK293 cells were cotransfected with 1 and 2 subunits alone or in combination with 1 or 2 subunits. Responses were evoked by application of 10 µM GABA. Representative traces are presented in Fig. 1A. Data are summarized in Table 1. In cells expressing 122 receptors, coapplication of 2 µM SEP-174559 enhanced GABA-evoked currents by 11 ± 3% (n = 4). Coapplication of 20 µM SEP-174559 produced a greater increase of 32 ± 4% (n = 11). Likewise, 2 and 20 µM (R,S)-zopiclone enhanced GABA responses by 34 ± 8% (n = 5) and 53 ± 11% (n = 11), respectively. In cells expressing 12 alone, neither SEP-174559 nor (R,S)-zopiclone had any effect up to 20 µM (n = 6). Also, no enhancement was seen for 1-bearing receptors. Rather, 121 receptors were modestly inhibited by 20 µM SEP-174559 by 9 ± 1% (n = 10). This inhibition of the 111 subtype was unexpected but not unique to cyclopyrrolones because 20 µM alprazolam and 20 µM diazepam also inhibited 111 currents by 83 ± 5 (n = 4) and 84 ± 7% (n = 4), respectively.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Subunit dependence of SEP-174559 modulation. Whole-cell currents were evoked by 10 µM GABA (open columns) in cells expressing recombinant GABAA subtypes. A, GABAA receptor 2-subunit is required for SEP-174559 modulation. Open columns depict the timing of GABA application. Filled columns depict the timing of coapplication of 20 µM SEP-174559. Scale is the same for all traces. B, modulation by SEP-174559 or (R,S)-zopiclone is not specific for receptors of a particular -subunit composition. Filled columns depict the coapplication of 20 µM SEP-174559 or 20 µM (R,S)-zopiclone. Note the rapid onset of potentiation by both drugs and relatively rapid recovery from potentiation after the removal of SEP-174559.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Effects of SEP-174559 on GABA dose-response curves. Whole-cell currents in cells expressing recombinant GABAA subtypes were evoked by various concentrations of GABA in the absence (control) or presence of 20 µM SEP-174559. Superimposed curve fits are given by the logistic equation I = Imax/(1 + (EC50/[agonist])nH). GABA EC50 and Hill coefficient (nH) values are given in Table 2. Data are mean ± S.E.M. for 4 to 12 cells/point.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   GABAA receptor modulation by zopiclone enantiomers. Whole-cell currents in cells expressing recombinant 122 receptors were evoked by 10 µM GABA. Open columns depict the timing of GABA application. Filled columns depict the timing of coapplication of 20 µM (R,S)-zopiclone or 20 µM (R)- or (S)-enantiomers. All reliably enhanced GABAA receptor currents to a similar extent; however, the slow reversal of (R,S)-zopiclone potentiation was mimicked only by (S)-zopiclone. Traces are from a single cell. Scale is the same for all traces.

Drug Actions on Other Receptors in Nicotinic Superfamily. Given their genetic and structural relatedness to GABA_A receptors, it was prudent to investigate whether SEP-174559 is active at nACh receptors or 5-HT₃ receptors. The activity of SEP-174559 was tested by whole-cell patch-clamp recording in transfected HEK293 cells expressing either 5-HT₃-A or 34 nACh receptors. SEP-174559 did not affect 5-HT₃ receptor currents. Responses were evoked by application of 100 µM 5-HT. Coapplication of 20 µM SEP-174559 did not alter the magnitude or decay time course of 5-HT-evoked currents (n = 4). SEP-174559 also did not affect responses evoked by 10 µM 5-HT (n = 3), which we determined is near the EC₅₀ for these receptors. Alprazolam (20 µM) also did not affect 5-HT₃ receptor currents evoked by 10 µM 5-HT (n = 2) or 100 µM 5-HT (n = 2). In contrast, SEP-174559 did inhibit nACh receptors currents. Responses were evoked by application of 1 mM ACh. Coapplication of 20 µM SEP-174559 reduced the magnitude of ACh-evoked currents by 35 ± 2% (n = 12) (Fig. 4). Similar inhibition by 34 ± 3% (n = 8) was produced by 20 µM (R,S)-zopiclone. Higher concentrations of both drugs (100 µM) further reduced ACh-evoked responses by 74 ± 2 (n = 7) and 71 ± 2% (n = 3), respectively. The mechanism of block by SEP-174559 and (R,S)-zopiclone was further examined and found to be noncompetitive in that the magnitude of inhibition was not dependent on agonist concentration (Fig. 5C). In three cells tested at various membrane potentials, the inhibition also was not voltage-dependent (Fig. 5, A and B). Together, these data suggest an allosteric mechanism of action by which drug binding affects channel opening or conductance rather than an open-channel blocking mechanism. Because such inhibition of nicotinic acetylcholine receptors (nAChRs) by benzodiazepine-site ligands has not been reported previously, it was important to determine whether this effect was unique to cyclopyrrolones. It was not. Coapplication of 20 µM alprazolam inhibited 1 mM ACh-evoked currents by 78 ± 3% (n = 4). Likewise, 2 and 20 µM diazepam inhibited ACh-evoked currents by 21 ± 2 and 94 ± 1% (n = 5), respectively, suggesting an inhibitory potency (IC₅₀) on the order of 5 to 10 µM under these conditions.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Nicotinic receptor inhibition by SEP-174559 and (R,S)-zopiclone. Left, whole-cell currents in cells expressing recombinant 34 nAChRs were evoked by 1 mM ACh. Right, whole-cell currents in cells expressing 5-HT3 receptors were evoked by 100 µM serotonin. Open columns depict the timing of agonist application. Filled columns depict the timing of coapplication of 20 µM SEP-174559 or 20 µM zopiclone.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Nicotinic receptor inhibition by SEP-174559 and zopiclone is noncompetitive. A, whole-cell currents in cells expressing recombinant 34 nAChRs were evoked by 1 mM ACh at various holding potentials. Open columns depict the timing of ACh application. Filled columns depict the timing of coapplication of 100 µM SEP-174559 or 100 µM zopiclone. B, whole-cell currents at various potentials are normalized to compare the magnitude and kinetics of inhibition by SEP-174559 and (R,S)-zopiclone. C, magnitude of inhibition was not dependent on the agonist concentration used to evoke the nAChR response. Inhibition was also produced by 20 µM alprazolam (ALPZ) and 20 µM diazepam (DIAZ), shown at the right. Data are mean ± S.E.M. for (n) cells as indicated above the columns.

Drug Actions on Receptors in Glutamate Receptor Superfamily. The activity of SEP-174559 at NMDA-type glutamate receptors was tested by whole-cell patch-clamp recording in cells expressing the NR1/2B receptor subtype. NMDA receptor currents were examined in Mg²⁺-free extracellular solution and stimulated by application of 100 µM glutamate plus 10 µM glycine. Coapplication of 20 µM SEP-174559 reduced the magnitude of agonist-evoked currents by 28 ± 4% (n = 5) (Fig. 6). Similar inhibition by 23 ± 5% (n = 5) was produced by 20 µM (R,S)-zopiclone. In contrast, SEP-174559 was inactive at AMPA- and kainate-type glutamate receptors. These were tested by examining glutamate-evoked responses in outside-out patches taken from cells expressing the GluR1 AMPA receptor or GluR6 kainate receptor subtypes. Responses were evoked by ultrafast application of 1 mM glutamate. Coapplication of 20 µM SEP-174559 after preexposure to the drug did not alter the peak amplitude or decay time course of either GluR1 (n = 3) or GluR6 (n = 3) receptor currents (Fig. 6).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   NMDA receptor inhibition by SEP-174559 and (R,S)-zopiclone. Left, whole-cell currents in cells expressing recombinant NR1/2B receptors were evoked by 100 µM glutamate plus 10 µM glycine. Open columns depict the timing of agonist application. Filled columns depict the timing of coapplication of 20 µM SEP-174559 or 20 µM (R,S)-zopiclone. Right, currents in outside-out patches taken from cells expressing recombinant GluR1 AMPA receptors or GluR6 KA receptors were evoked by 1 mM glutamate in the absence or presence of 20 µM SEP-174559.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The actions of SEP-174559 were examined at a variety of neurotransmitter receptor ion-channel subtypes. As expected, a benzodiazepine-like enhancement of GABA_A receptor currents was confirmed. The facilitation of GABA_A responses by SEP-174559 required the presence of the 2 subunit, which is prevalent in vivo (Laurie et al., 1992; Wisden et al., 1992; Fritschy and Mohler, 1995) and accounts for most high-affinity benzodiazepine binding (Gunther et al., 1995). In contrast, SEP-174559 was not specific for any particular  subunit. Rather, SEP-174559 was broadly active at 1-, 2-, and 3-bearing receptor subtypes. Estimated K_d values for these receptors were all in the range of 1 to 6 µM, and the mechanism of action of SEP-174559 at GABA_A receptors was found to involve an allosteric enhancement of the affinity for GABA binding as has been proposed for other benzodiazepine-site ligands.

It is perhaps not surprising that modulation by (R,S)-zopiclone was also not -subtype specific. Indeed, (R,S)-zopiclone modulation has been demonstrated for the 122 subtype but also for 22 absent an  subunit (Im et al., 1993), suggesting the  subunit is not important for (R,S)-zopiclone modulation. Yet, paradoxically, (R,S)-zopiclone binding has been shown to involve the -subunit His101 residue, which is a necessary component of the benzodiazepine binding site (Davies et al., 2000). Likewise, our studies also indicate at least some involvement of the  subunit in (R,S)-zopiclone binding because K_d estimates from kinetic analyses of 1-, 2-, and 3-bearing subtypes differed by as much as 7-fold between 1-bearing (highest affinity) and 3-bearing (lowest affinity) subtypes. The present study also confirms that (R)- and (S)-zopiclone enantiomers are both active at GABA_A receptors, whereas (S)-zopiclone has relatively higher affinity and likely accounts for much of the action of racemic zopiclone.

In general, SEP-174559 and (R,S)-zopiclone had similar effects on the various receptor subtypes tested. However, in all measures of 1-, 2-, and 3-bearing GABA_A receptors, the enhancement produced by (R,S)-zopiclone was consistently greater than that produced by SEP-174559 at equimolar concentrations, and kinetic analyses indicated a 5- to 10-fold higher apparent affinity (lower K_d) of these receptors for (R,S)-zopiclone compared with SEP-174559. The relatively greater enhancement by (R,S)-zopiclone might reflect its higher affinity for GABA_A receptors or a "superagonist" action at the benzodiazepine site as has been suggested previously (Davies et al., 2000). However, because no enhancement is seen at saturating GABA concentrations, it is likely that these drugs differ only in their affinity for binding to GABA_A receptors. Thus, the fact that these drugs have similar actions raises two important questions regarding their in vivo effects: 1) might some of the actions of (R,S)-zopiclone in fact result from its metabolites, including SEP-174559? and 2) why is SEP-174559 the more potent and selective anxiolytic agent? Unfortunately, the present study does not answer these questions except to demonstrate the lack of  subtype specificity of both drugs. Satisfactory answers will require additional pharmacokinetic and pharmacological studies.

With regards to the first question, the available data suggest that (R,S)-zopiclone and SEP-174559 have similar molecular mechanisms of action. Given the relatively greater potency of (R,S)-zopiclone compared with SEP-174559, it seems unlikely that the lower affinity metabolite could account for the anxiolytic or anticonvulsant effects of (R,S)-zopiclone in vivo without producing similar impairments in locomotor activity or motor coordination (Carlson et al., 2001). However, in relating drug affinities in vitro to effective doses in vivo, one should like to know something about the bioavailability, plasma concentrations, and brain penetration of the drugs in question. In this regard, zopiclone is rapidly absorbed, distributed in various tissues, including brain, and achieves plasma concentrations of 30 to 90 ng ml¹ (100-300 nM) after therapeutic doses (Gaillot et al., 1983), which is comparable with its effective concentration at GABA_A receptors in vitro (Table 2; Reynolds and Maitra, 1996; Davies et al., 2000). There are as yet no comparable pharmacokinetic data on SEP-174559, but it may be important to consider that SEP-174559 is far more soluble in aqueous solution than zopiclone or benzodiazepines and so might differ dramatically in its ability to act at central GABA_A receptors. If so, this could help to explain why two drugs with apparently 10-fold different affinities are generally effective at similar doses, but cannot explain the apparent selectivity of SEP-174559 in behavioral studies to produce anxiolytic and anticonvulsant effects without also producing sedation as does (R,S)-zopiclone (Carlson et al., 2001).

This bears on the second question. Benzodiazepines are thought to exert their sedative and anticonvulsant effects through 1-bearing GABA_A receptors (Rudolph et al., 1999; McKernan et al., 2000) and their anxiolytic effects through 2-bearing receptors (Low et al., 2000). Clearly, SEP-174559 is not selective for 2-bearing GABA_A receptors. Neither is (R,S)-zopiclone. Both drugs are active at 2-bearing receptors, but also at 1- and 3-bearing receptors. Based on our current understanding then, both drugs would be expected to produce anxiolytic effects (via 2), anticonvulsant effects (via 1), and sedation (via 1). (R,S)-Zopiclone fulfills all of these expectations, whereas SEP-174559 fails to produce sedation except at very high doses (50- to 100-fold higher than required for anxiolytic or anticonvulsant effects) (Carlson et al., 2001). Together, these observations suggest that other (i.e., not 1-, 2-, nor 3-bearing) receptors must be involved in the sedative effects of benzodiazepine ligands. These effects might involve other GABA_A subtypes or receptors outside the GABA_A receptor family. In addition to the commonly appreciated enhancement of GABA_A receptors, our studies indicate that SEP-174559, (R,S)-zopiclone, and other classical benzodiazepines inhibit 1-bearing GABA_A receptors, nACh receptors (34 subtype), and NMDA receptors (NR1/2B subtype). Such inhibitory effects have not been reported previously, and their significance to the main effects or side effect profiles of benzodiazepine-site ligands is unclear. These actions are relatively low potency. However, it may be prudent to consider whether these or other similar actions might be related to the sedative, amnesic properties, or addictive potential of benzodiazepine ligands. As such, it may be informative in future drug development efforts at least to assess the actions of benzodiazepine-site ligands at nACh and NMDA receptors.