TiPS - December 1992 [Vol. 131

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GABA* receptors: ligand-gated Cl- ion channels modulated by multiple drug-binding sites Werner Sieg hart GABA, receptors are ligand-gated Cl- ion channels and the site of action of a variety of pharmacologically and clinically important drugs. In this review evidence is summarized indicating that these drugs, by interacting with several distinct binding sites at these receptors, allosterically modulate GABA-induced Cl- ion ftux. Other results indicate that the affinity, as well as the modulatory efficacy of drugs, changes with receptor composition. A thorough investigation of the pharmacological properties of the individual binding sites on difjkent GABA, receptor subtypes could open new avenues for selective modulation of GABAA receptors in different brain regions.

GABAA receptors of the vertebrate nervous system are ligand-gated Cl- ion channels and the targets of a variety of pharmacologically and clinically important drugs. Thus, binding studies and electrophysiological and behavioral experiments indicated that the anxiolytic, anticonvulsant, muscle relaxant and sedativ+hypnotic benzodisome anxiogenic or azepine&, convulsant g-carbolineszA, some depressan t barbiturates’~*4, some anesthetics (such as etomidate3, propofo15 or alfaxalone4,“‘), some anxiolytic, anticonvulsant and hypnotic steroid&‘, some convulsants (such as bicuculline or picrotoxinin3,4) and some anthe!mintic and insecticidal compounds (such as avermectin Bi, [Refs 3,8] or lindanep produce at least part of their pharmacological effects by interacting with GABAA receptors. A large series of experiments indicated that, in most cases, the above-mentioned compounds do not interact directly with the GABA binding site, but exert their action by binding to additional allosteric sites at GABAA receptors (Fig. 1). This overview briefly outlines evidence for the existence of these allosteric binding sites. For more detailed information on these sites, consult the references cited herein.

W. Sieghart is Associate Professor and Chief at the Department of Biochemical Psychiatry, University Clinic fvor Psych&y, Wiihringer Giirtel M-20, A-1090 Vienna, Austria. @ IWZ.UsevierScience

Publishers

Ltd (UK)

The GABA-binding site of GABA, receptors GABA, by binding to GABAA receptors, increases the neuronal membrane conductance for Clions, resulting in membrane hyperpolarization and in reduced neuronal excitability1,4. This effect can be competitively inhibited by bicuculline4. The GABA* binding site can be selectively labeled by agonists like [3H]GABA or [3H]muscimo13*7. This site shows both high and low affinity for GABA and its agonists, with & values in the nanomolar or micromolar range, respectively. The low-affinity GABA* recognition site seems to be an antagonist-preferring site’, since it can be selectively labeled by specific antagonists, such as (+)-bicuculline or SR95531. Both the lowand high-affinity forms of the GABA* binding site show similar drug specificity, and pentobarbital increases the number of high-affinity sites at the expense of low-affinity sites. Thus, highand low-affinity GABA* binding sites may represent different conformational states of the same receptor (for review see Ref. 7). It is now generally assumed that GABA exerts its physiological effect by acting at the low-affinity binding sites’. Thus, it has been demonstrated that micromolar concentrations of GABA, or its analogs, are necessary to activate the Cl- channel in electrophysiological experimentslO, and to modulate other binding sites at the GABAA recepto$“. The high-

affinity GABAA sites might, thus, represent a desensitized form of the GABA* receptor. In addition to the GABA* agonists GABA and muscimol, which maximally activate Clconductance, some other compounds have been identified which seem to act as partial agonists at the GABA binding site3. In spite of some evidence for possible heterogeneity of the GABA binding site of GABA* receptors3, so far no compound has been identified which selectively interacts with a subtype of the GABA* binding site. The benzodiazepine-bindiig site of GABA,, receptors Electrophysiological experiments have indicated that benzodiazepines, such as diazepam or fhmitrazepam, enhance the actions of GABA at the GABA* receptor by increasing the frequency of Clchannel opening*. Biochemical experiments demonstrated the existence of specific high-affinity binding sites for benzodiazepines on brain membranes which are closely associated with GABA, receptors3. Thus, . [3H]fl~itrazepam to EYZrerZ branes was stimulated by GABA or muscimol, and this stimulation was inhibited by (+)-bicuculline3. Reciprocally, benzodiazepines increased the binding of GABA to GABA,+, receptors”. Since there was an excellent correlation between the clinical potency of benzodiazepines and their affinity for this [3H]fl~trazepam binding site, it is assumed that these binding sites (‘central benzodiazepine receptors) are the pharmacological receptors by which the benzodiazepines exert their clinically important actions’. Other (‘peripheral’) benzodiazepine binding sites are localized on the outer mitochondrial membrane of many tissues, including brain, are pharmacologically distinct from, and unrelated to, these GABA, receptor-associated benzodiazepine binding sites13. Whereas most of the classical benzodiazepines, such as diazepam or flunitrazepam, seem to exhibit a similar affinity for benzodiazepine receptors in different brain tissues, several benzodiazepine and non-benzodiazepine compounds have been identified which can distinguish

TiPS - December 1992 [Vol. 731 between at least two different GABA* receptors containing distinct benzodiazepine binding sites” (BZ, or BZz). Other experiments indicated that in addition to benzodiazepine receptor agonists which enhance the GABA-induced Clion flux, other compounds exist (‘inverse benzodiazepine receptor agonists’) which by interacting with the benzodiazepine binding site reduce the GABA-induced Clion flux*. A third group of compounds interacting with the benzodiazepine site of GABAA receptors (‘benzodiazepine receptor antagonists’) in most cases does not influence GABA-induced ion fluxes but antagonizes the actions of benzodiazepine receptor agonists or inverse agonists*. Recently, evidence has accumulated indicating that some esters of g-carboline-3-carboxylate15*16 or the anxiolytic cyclopyrrolones zopiclone and suriclonelr, ligands which ori@nally were thought to interact with the benzodiazepine binding site of GABA,, receptors, might bind to regions or domains of this site different from those interacting with benzodiazepines. These regions may either overlap or be entirely discrete. The possible identity of the g-carboline and suriclone binding domain so far has not been investigated. The piaotoxikn/TBPS-binding site of GABA* receptors Picrotoxinin and some bicyclic cage compounds are convulsants which antagonize GABA-induced Clconductance responses3,*. These compounds, however, did not inhibit GABA receptor binding and did not displace benzodiazepines from their highaffinity binding sites3. Binding sites identified by [3H]ar-dihydropicrotoxinin (DHP)3 or the cage convulsant [35S]t-butylbicyclophosphorothionate (TBPS), which exhibits a better signal-to-noise ratio than 13H]DHP (Ref. ll), seem to be closely associated with the Cl- ion channel of the GABAA receptor. Convulsant compounds that bind to the DHWTBPS site seem to reduce directly Cl- conductance by sterically hindering the entry of Cl- across the ion channel (for review see Ref. 6). GABA and compounds which mimic or facilitate the effects of the GABA,+,

447

avermectin

picrotoxinin-TBPS

Fig. 1. Allosteric binding sites of GABA, receptors. Additional binding sites might exist for propofol, clomethiezofe and ethanol. TBPS, &botytbicy&phomtbionate.

receptor (e.g. benzodiazepines, barbiturates, steroids, see later), allosterically inhibited [35S]TE%PS binding by reducing its binding affinit$. Compounds reducing the efficacy of GABA at GABA,+ receptors, such as some convulsant g-carbolines enhanced [35S]TBPS binding affinity through specific interactions with the benzodiazepine receptor. Thus, the highaffinity TBPS binding might be associated with the ‘closed’ conformation of the Cl- ion channeP. The barbiturate-binding site of GABA* receptors Sedative hypnotic barbiturates, such as pentobarbital or secobarbital, in electrophysiological studies enhance the actions of GABA by increasing the mean channel open time1s4. At higher concentrations, barbiturates are able to enhance Cl- conductance in the absence of GABA4. No direct binding of barbiturates to their recognition sites has been performed due to their low affinity for these sites. However, the interaction of barbiturates with GABA* receptors was indirectly investigated. Barbiturates enhanced GABA, receptor and benzodiazepine receptor affinities3 in a manner that correlated with their order of potency as anesthetics and hypnotics. In addition, these bar-

biturates inhibited the binding of [3WDHP or [35S]TBPS again in the ranic order of potency as hypnotics3*‘1. Whereas convulsants, such as picrotoxinin, TBPS and pentetrazole, but also some convulsant barbiturates inhibited TBPS bindcompetitively, depressant Ebiturates (oentobarbital, secobarbital) and ielated compounds, such as etazolate and etomidate, seem to interact allosterically with the [%]TBPS binding sitesra**‘_ This seems to indicate that the depressant barbiturates enhance GABA-induced Cl- ion fhrx by interacting with a binding site which is different from that for GABA agonists, for benzodiazepines or for DHP/TBPS. As with the GABA or benzodiazepine site, partial agonists seem to exist for the barbiturate binding site3. In addition, the pharmacological action of barbiturates on GABA, receptors differed in different brain tissues, possibly indicating the existence of GABA* receptor subtypes with different barbiturate binding properties3. The steroid-binding site of GABA* receptors Several steroids, such as the anesthetic alfaxalone (5~pregnan3at-ol-11,20-dione) or the sedativehypnotic and anxiolytic Jar-hy-

448 dmxylated, ~~r&uced metabolites of progesterone and desoxycortone, like barbiturates’, were found to enhance GABA-s~~rn~at~ Clconductance in rat brain by prolonging the open time of the Clchannels. In addition, these compounds enhanced the binding affinity of the GABA, agonist @-@nuscimd or of the benzodiazepine receptor agonist [3H~flunitmzepam, and inhibited binding of [ssS]TBPS to the receptor (for review see Refs 6 and 7% other experiments indicated that barbiturates potentiated steroid-activated bansmembrane curren@, and, in studies investigating [35s]TBPS or [3H)flun&razepam binding, interacted with stxoids in a manner inconsistent with competition at a common site (rOr review see Ref. 6). These experiments provided evidence for a separate site of action of steroids which is distinct from the binding sites for GABA, benzobarbiturates and diazepines, DHP/TBPS. The highly hpophihc nature of the steroids and the evidence that phosphohpids are capable of binding steroids with high specificity, raise the possibility that their effects are mediated by specific interactions with the membrane-lipid GABA_&receptor protein interfac@, However, the stringent structural requirements and the nanomolar potencies of steroids in the presence of GABA argue in favor of a specific action at the receptor proteins. The avermectin Bl,-binding sitr?af

GABA, we@ws Avermeetin B1, (AVM) is a macrocyclic lactone from Str@omyces ane?7&lis with potent insecticidal and anthelmintic actions~-= ~e~~phys~o~og~c~ studies demonstrated that AVM and its structural analogs increase the permeability of vertebrate and invertebrate nerve and muscle membranes to Cl- ions. In most of these cases, avermectins exhibited either synergistic or antagonistic interactions with exogenously applied GABA, and at least part of the avermectin-dependent increase in a- conductance was reversed by the application of GABA, receptor antagonists*‘. Other studies indicated that there is a high-affinity binding site for [sH]AVM on brain membranes and that this binding

site exhibits a series of complex allosteric interactions with binding sites for GABA, for benzobarbiturates or diazepines$ TBPS~,~~~.Thus, depending on the conditions concentration and used8 AVM stimulated or inhibited high-affinity 13HIGABAor f3H]&nitrazepam binding. AVMstimulated [%$PWS binding and high-affinity [3HjAVM binding was modulated by GAB& receptor agonists and antagonists in a Cl- ion-dependent ways=. These results indicated a close assoeiation of AVM binding sites with the GABA, receptor complex” and suggested that these AVM sites are not identical with the GABA, the benzodiazepine, the barbiturate or the picrotoxininTBPS binding sites of this Irrceptors*22. The relationship of the AVM binding site to the steroid or Ro.54864 binding site {see later) so far has not hem investigated.

The Ro54864-binding site of GABA& receptors R05.4864~the 4’-chlom-derivative of diazepam~ at nanomolar concentration is a prototypic ligand for the ‘peripheral’ benzodiazepine binding site13. At micromolar levels, however, this compound intmacts with the GARA, receptor. Ro.54864 is a potent convulsant, and its convulsant effect is antagonized by barbiturates, diazepam and other clinically useful benzodiazepines?. In el~~phys~olo~c~ experiments, Ro54864 inhibited GABAstimulated Cl- flux and enhanced neuronal firing induced by TBPS23. In binding studies* it did not interact with the GABA or benzodiazepine binding site of GABAA receptors, but it enhanced the binding of [35S]TBPS, and this effect was modulated by GABA acting at GABAA receptors2’“. Coltectively, the evidence points to a unique and relatively low-affinity (Kd 250 n&t) Ro54864 site that is linked to a GABAA receptorz4. In addition to Ro54864, other compounds such as the phenylquinolines PK8165 and PK9084 and the isoquinoline carboxamide derivative PK11195 seem to modulate GABA,+ receptors by binding to the Ro54864 sitez*. The cornpound PK11195, which at subnanomolar concentrations specifically binds to the peripheral benzodiazepine binding site13, at

micromolar concentrations blocked the effects of Ro54864 on [35S]TBPS binding and potentiated the electrophys~o~ogi~al effects of the GABAA agonist muscimd, indicating that this compound exhibits actions opposite to those of Ro54864 (Ref. 24). The Zn2’-binding s&eof GABAA

receptors

Zn2+ is concentrated in synaptic terminals and released with electrical activity in sufficient quantities to play a potential role in neurotransmission26. Moreover, Zn*’ and, to a lesser extent certain other metal cations such as Cd”, Ni*“, Mn2+ and Co”, inhibited the GABA response of neurons in a variety of organisms; whereas Ca2’, M$+ or Ba*+ were consistently without effect when applied extracellularly~7. Heat-inactivation studies2s and radioligand binding studiesz’ suggested the presence of a Zn*’ binding site at some, but not all, GABAA receptor subtypes (see Box). This site seems to be localized extracelhrlarfy and to be distinct from the GABA, the benzodiazepine, barbiturate, picrotoxinin and steroid recognition sites27.

The intera&m of CT wit& GABAh receptors

Since modulation of GABAA receptors influences GABAinduced Cl- ion flux, a modulation by Cl- of the various binding sites at the GABAA receptor is not surprising. Thus, evidence has accumulated indicating a dependence on, or a strong modulation by, Cl-, P;r-, I-, NO,-, SCN- or CQof most if not all binding sitesF and allosteric interactions between bindin sites of GABAA receptors3”“r1*,* P. The specificity of the anion modulation of the various binding sites was proposed to represent coupling of these sites to a Cl- ion channel.

Other possible binding s&s of GAlBAAreceptors

Both pharmacological and electrophysiological evidence suggest that the anxiolytic, anticonvulsant and sedative-hypnotic cIo%ethiazole3ad3E or the intravenous general anesthetic propofol (2,6-diisopropylphenol)5*32 may exert at least some of their actions through the GABAA receptor complex, Thus* dlo-

Allosteric modulatory sites on recombinant GAlBAA receptors M&?c&r cloning studies have provided evidence for the existence of at least six e-y_, four @-, three y-, one 8-

and two p-subunits of the GABAA receptor. Each of these subunits exhibits a distinct regional distribution in the brai@. Currently, it is assumed that five subunits are necessary for the ovation of GABA-induced Cl- ion channels. Depending on the number and types of different subunits forming the GABAAreceptor, several hundred to several thousand structurally different combinations are possible’. Presumably, only a small part of these theoretically possible subtypes is actually used in the brain. Unfo~nately~ the exact subunit ambition of not even one i,ative GABA* receptor subtype is known at present. So far, only a limited number of GABA* receptor subunits or subunit combinations have been expressed in Xenopus oocytes or in cell culture systems and the presence of allosteric modulatory sites on the resulting recombinant receptors has not been systematically investigated. In addition, evidence indicating that at least some properties of recombinant receptors may differ, depending on the type of expression system used, has accumulatedL*3. Thus, not all the resuits obtained so far can be generalized for receptors present in the brain. Expression studies indicated that most, if not all, GABAAreceptor subunits, although rather inefficiently, are able to form homooligomeric ion charmeis, at least some of which can be activated by GABA and inhibited by bicuculline. These channels exhibited multipIe conductance states and showed desensitizationla. Channels containing two different subunits formed more efficiently, could be activated by lower GABA concentrations, and the induced Cl- ion fluxes were higher than in homomeric ehatutels~*. However, cooperativity of GABA in gating the channel was absent in most dual subunit combinations tested43. A coexpression of (Y-, fi- and yz-subunits resulted in large GABA-gated Cl- currents which could be inhibited by bicuculline. Cooperativity was apparent in most of these receptors and, as with receptors containing two different subunits, the GABA responsiveness and the conductance properties were dependent on the subunit combination investigated4,‘. Similar to the GABA binding site, binding sites for picrotoxinin~barbiturates and possibly steroids seem to be formed by the assembly of most GABAA receptor subunits. Thus, GABA-induced Cl- ion fhzx of homooligomeric channels or channels containing two or three different subunits was consistently inhibited by picrotoxinin and enhanced by barbiturates’. Enhancement by steroids so far has been investigated only in @Isubunit containing homo- and heterooligomeric channels6. The efficacy of steroids to enhance GABAinduced Cl- ion flwc in heterooligomeric channels depended on the dual or triple subunit combination investigated’. A ZnZ+binding site seems to be present on some, but not all, homo- or heterooligomeric channels. Thus, channels which contained only arl or 82 or a combination of these subunits were blocked by Zn2+, whereas channel& which contained the y2-subunit alone or in combination with 1y- or/and @subunits were rather ‘nsensitive to Zn*” (Ref. 8). At least two different subunits, one of which is a YIor a y2-subunit, however, seem to be necessary for the formation of Ro54864 binding sites’,“. Thus, R054864 inhibited GABA-induced ion flw in all recombinant

receptors containing yl- or y2-subunits in dual or triple combinations with a+ o&and @-subunits. This compound, however, was inactive at channels composed of al- and #l+ubunits. Preliminary evidence indicates that propofol11 or clomethiazole’2 are able to enhance GABA-induced Q- Bux in Chinese hamster ovary cells co-transfected with arl- and &-subunits. Other evidence seems to indicate that only the longer, but not the shorter, alternatively spliced variant of the yz-subunit, when coexpressed with al- and &-subunits, results in GABAinduced Cl- ion fluxes which are enhanced by ethan01~. The possible presence of avermectin B1, binding sites on recombinant receptors has not been investigated so far. Channels produced by expression of only one or two different GABAAreceptor subunits usually showed no, or an atypical, response to benzodiazepine receptor ligands*s. A robust modulatisn by benzodiazepine receptor ligands was genera& observed, however, when or-, fl- and y2-subunits were coexpressed in a single cellL43. These and other results mentioned earlier, indicate that recombinant receptors contain@ [Y-, & and yz-subunits most closely resembie GABAA receptors found in the brain. Depending OR their subunit composition, recombinant GABA* receptors exhibited different benzodiazepine bindilg pmperties. Especially, the type of the [Y- or y-subunit strongly influenced not only the affinity of various selective and nonselective benzodiazepine receptor ligands for the receptorl-3 but also the efficacy of benzodiazepine receptor ligands for the enhancement of GABA-induced Cl- ion fh~+~~~*. So far, no firm conclusion can be drawn on the pharmacological properties of GABAA receptors containing the &subm@. However, the GABA-induced Ci- ion flux of receptors containing the pl-subunit could be inhibited by picrotoxkin but not by bicuculline. In addition, this GABA-effect was not modulated by pentobarbital or by benzodiazepine receptor &and% Recombinant receptars containing pl-subunits thus resemble GAB& receptors16. References

1 Burt, D. R and Kamatchi,G. L. (1991) FASEBJ. 5,291~2923 2 Liiddew, H. and W&den,W. (1991)TrendsPharmacol.Sci. II&@-51 3 Doble, A. and Martin. I. L. (1992) TrendsPkamxof. Sci. 23, 76-81 4 Sigel, E., Baur, R., Tmbe, G., M&ler, H. and Malberbe,P. (1990) Neuron5,703-711 5 Knoflach,F. ef al. (1992) Eur. J. Neurosci. 4, l-9 6 Puia, G. et nl. (1990) Neuron4,75+7# 7 Lan, N. C., Gee, I.USAk9; 3620-3624 15 Benke, D., Mertens, S., Tneciak, A., Gillessen. D. and Miihler,H. (1991) FEBS L&f. 283,145-149 16 Shimada, S., Cutting, G. and Uhl, G. R. (1992) MUI. Phurmacol,41,683-687

TiPS - December 1992 CVol.231 methiazole, as well as pnzpofo], potentiated dose-dependently GABA-activated currents, and at higher doses directly activated the GABA, receptor by increasing the Cl- conductance of the cell membranes in a bicuculline-sensitive way. Clomethiazole as well as propofol was found to inhibit allosterically the binding of [%]TBPS or to enhance the binding of [%I]muscimol but had no effect on [3H]flunitrazepam or on pentobarbital-enhanced [3H]-flunitrazepam bindingae3. These findings seem to indicate that these compounds can perturb the GABA* receptor complex by interacting with a site distinct from other sedatives-hypnotics such as barbenxodiazepines or biturates, FlgOlliStS. Additional GABA, studies, however, are necessary to clarify further the mode of action of domethiazole and propofol and their possible interaction with the same binding site. Ethanol shares some properties with barbiturates and benzodiazepines since it exhibits anticonvulsant, anxiolytic and sedative activity, and the development of cross-tolerance among these compounds has also been observed= Ethanol potentiated GABA-mediated 36cl- transport into cultured spinal neuron? and rat brain synaptoneurosomes35. However, electrophysiological data are controversial. In some systems, ethanol potentiated GABA-induced responses, in other systems this effect was not observed=. These and other studies (see Box) indicate an interaction of ethanol with at least some GABAA receptor subtypes.

cl

cl

0

Most of the evidence summarized here was obtained from experiments using intact tissue or brain membranes. However, the recent cloning of GABAA receptors and their multiple subunits supports the existence of a large variety of different GABAA receptors in the brain (see Box). Thus, results from pharmacological experiments using intact brain tissue or brain membranes represent the average properties of a variety of different GABA* recep-

tors. Properties of indiviuual receptors so far can only be investigated in studies using recombinant receptors. A careful investigation of not only the GABA, benzodiazepine or steroid binding site, but also of other less known allosteric sites of GABAA receptors will be necessary to characterize fully recombinant receptors containing defined subunits. Not all of the various binding sites might be present on each GABAA receptor subtype (see Box). The properties of the individual allosteric sites might vary, and it is quite probable that the direction in which compounds influence GABA-induced Cl- ion flux will change with different receptor composition. Thus, for instance, evidence is accumulating that benzodiazepine receptor agonists behave as partial agonists, and inverse agonists behave as agonists when the subunit composition of the GABAA receptor is changed (see Box). In addition, not only the pharmacology of the allosteric binding sites but also that of the GABA binding site is dependent on the subunit composition. Subunits and subunit combinations seem to exist which produce GABA receptors insensitive to bicuculline (see Box). These receptors more closely resemble GABAc than GABAA receptors. Thus, the identification of GABAA receptor subunit combinations actually occurring in the brain, the investigation of their pharmacological properties and their regional distribution might not only explain contradictory results obtained in different brain tissues, but also could open new avenues for the selective modulation of GABA* receptor subtypes in distinct brain regions. References 1 Study, R. E. and Barker, J. L. (1981) Proc.

Nat1 Acad. Sci. USA 78,7180-7184 2 Haefely, W., Kyburz, E., Gerecke, M. and Mohler, H. (1985) Adv. Drug Res. 14, 167-322 3 Olsen, R. W. (1982) Anna Rev. Pharmacol. Toxicol. 22, 245-277 4 Bormann, J. (1988) Trends Neurosci. 11, 112-116 5 Hales, T. G. and Lambert, J. J. (1991) Br. J. Phartaacol. 104,619-628 6 Gee, K. W. (1988) Mol. Neurobiol. 2, 291-317 7 Schumacher, M. and McEwen, B. S. (1989) Mol. Neurobiol. 3,275-304 8 Drexler, G. and Sieghart, W. (1984)

Neurosci. Lett. 50, 273-277 9 Lawrence, L. J. and Casida, J. E. (1984) Life Sci. 35,171-178 10 Segal, M. and Barker, J. L. (1984) J. Neurophysiol. 51, 50&515 11 Squires, R. F., Casida, J. E., Richardson, M. and Saederup, E. (1983) Mol. Pharmacol. 23,326-336 12 Skeritt, J. H., Willow, M. and Johnston, G. A. R. (1982) Neurosci. Mt. 29,63-66 13 Verma, A. and Snyder, S. H. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 307-322 14 Sieghart, W. and Schlerka, W. (1991) Eur. J_ pharmacol. 197,103-107 15 Chiu, T. H. and Rosenberg, H. C. (1985) J. Neurochem. 44,306-309 16 Dellouve-Courillon, C., Lambolez, B., Potier, P. and Dodd, R. H. (1989) Eur. J. PharnwoJ. 166,557562 17 Trifiletti, R. R. and Snyder, S. (1984) Mol. Pharmacol. 26,458-469 18 Supavilai, P. and Karobath, M. (1984) J. Neurosci. 4,119~1200 19 Ticku, M. K. and Rastogi, S. K. (1986) in Molecular and Cellular Mechanisms of Anesthetics (Roth, S. H. and Miller, K. W., eds), pp. 179-188, Plenum 20 Peters, J. A., Kirkness, E. F., Callachan, H., Lambert, J. L. and Turner, A. J. (1988) Br. 1. Pharmacol. 94,1257-l269 21 Payne, G. T. and Soderlund, D. M. (1991) J. Biochem. ToxicoJ. 6,283-292 22 Drexler, G. and Sieghart, W. (1984) Eur. J. Pharmacol. 101,201-207 23 Dai, K-S. and Woolley, D. E. (1991) Brain Res. Bull. 27, 13-17 24 Gee, K. W. (1987) J. Phnrmacol. Exp. Ther. 240,747-753 25 Gee, K. W., Brinton, R. E. and McEwen, B. S. (1988) J. Pharmacol. Erp. Ther. 244, 379-383 26 Xie, X. and Smart, T. G. (1991) Nature 349,521-524 27 Celentano, J. J., Gyenes, M., Gibbs, T. T. and Farb, D. H. (1991) Mol. Phnrmucol. 40,766-773 28 Squires, R. F. and Saederup, E. (1982) Mol. Pharmacol. 22,327-334 29 Mackerer, C. R. and Kochman, R. L. (1978) Proc. Sot. Exp. Biol. Med. 158, 393-397 30 Moody, E. J. and Skolnick, P. (1989) Eur. J. Phortnacol. 164,153-158 31 Hales, T. G. and Lambert, J. J. (1992) Eur. J. Pharmacol. 210,239-246 32 Concas, A., Santoro, G., Serra, M., Sanna, E. and Biggio, G. (1991) Brain Res. 542,225-232 33 Nakahiro, M., Arakawa, 0. and Narahashi, T. (1991) J. Pharmacol. Exp. Ther. 259,235-240 34 Mehta, A. K. and Ticku, M. K. (1988) 1. Pharmacol. Exv. Ther. 246.558-564 35 Suadak, P. d., Schwartz, R. D., Skolnick, P. and Paul, S. M. (1988) Bruin Res. 444,340-345

PK11195: 1-(2-chlorophenyl)-N-methyl-N(l-methyl-propyl)3-isoquinaline carboxamide PK8165: phenyl-2[(piperidinyl-4)-2-ethyl]4-quinoline PK9084: phenyl-2[(piperdinyl-3)-Z-ethyl]4-quinoline Ro54864r 7-chloro-1,3-dihydro-l-methyl-5(p-chlorophenyl)-Z&1,4-benzodiazepine2-one SR95531: 2-(carboxy-3’-propyl)-3-amino-6p-methoxyphenyl-pyridazinium bromide