A Functional Comparison of the Antagonists Bicuculline And

A Functional Comparisonof the Antagonists Bicuculline and Picrotoxin at Recombinant GABAAReceptors

B. J. KRISHEK,l S. J. MOSS2 and T. G. SMARTl*

‘TheSchool of Pharmacy,Departmentof Pharmacology,29–39BrunswickSquare,London WCIN IM, U.K. and 2Departmentof PharmacologyandMRC LMCB Universi~ College, Gower Street, London WCIE 6BT, U.K.

(Accepted2 July 1996)

Summary-AllostericmodulationofGABA~receptorfunctionby a numberofligandshasbeenshownto be dependentonthesubunitcompositionofthereceptorcomplex.Inthisrespect,modulationofGABA~receptors bytheantagonistsbicucullineandpicrotoxinwasexaminedinXenopus laevis oocytesexpressingrecombinant GABA~receptorscomposedof combinationsof murineal, /?1,y2S and y2L subunits.Bicucullineand picrotoxinreducedGABA-activatedresponsesmediatedby GABA~receptorscomposedof ulj?l, ulflly2S and121flly2Lsubunitsina dose-dependentmanner.GABAequilibriumconcentration–responsecurvesforeach receptorconstructwere shiftedto the rightby increasingconcentrationsof bicucullinein a competitivemanner, whereas picrotoxin induced a slight lateral shift as well as a depressionof the maximum response consistent with a mixed/non-competitiveinhibitorymechanism.GABAconcentration–responsecurvesin the absenceand presenceof bicucullinewere subjectedto Schildanalysis,which revealedsimilar pK~values of approximately 5.9 for alfll, ctl/31y2Sand ctl/lly2L receptorconstructs.Concentrationinhibitioncurveswere used to estimate IC50values forpicrotoxinwhichapproximatedto 0.4-0.6 MM.The apparentpK~values for bicucullineand the IC50Sfor picrotoxin were relatively unaffected by the GABA~ receptor isoforms used in this study, and in particular, by the absence of the y2 subunitin the alfll GABAAreceptor complex.The similarity of the pK~s reported in this study to those previously reported using native neuronal preparations, which are likely to represent heterogeneousGABA~ receptor populations, further indicates the lack of dependence on receptor subunit compositionfor the inhibitoryaction of bicuculline. Copyright0 1996Elsevier Science Ltd.

Keywords—RecombinantGABA~receptors,bicuculline, picrotoxin,Xenopus oocyte.

y-Arninobutyric acid (GABA) is the major inhibitory GABAAreceptors are known to possess a variety of neurotransmitter in the mammalian central nervous allostericbindingsitesfrom which a numberof drugscan system (CNS), a role that is reliant upon the almost modulatereceptorfunction(Macdonaldand Olsen, 1994; ubiquitous distribution of “type A“ GABA receptors. Smart, 1996). Previous studies, utilizing recombinant Molecularcloning studies have establishedthat GABA~ GABAAreceptors, have established that some pharma- receptors are hetero-oligomeric proteins composed of cological properties are critically dependent on the assemblies of different subunit polypeptides. These subunitcompositionpresentwithin the receptorcomplex. subunits are characterized into four discrete subunit For example, receptors composed of ctlfll or alf12 families based on amino acid sequence identities. The subunits are substantially inhibited by zinc ions com- majority of these subunit families possess multiple pared to ul~ly2S or ctlf12y2Ssubunit combinations members, designated as: ct(l-6), ~(14), y(14) and (Draguhnet al., 1990;Smart et al., 1991, 1994).Further- d(l) (Rabow et al., 1995; Sieghart, 1995). Pharmacolo- more, the y2 subunit has been shown to be important in gical and physiological properties observed for native conferring a sensitivity upon the GABAA receptor to neuronal GABAA receptors are most frequently repro- modulation by benzodiazepines (Pritchett et al., 1989; duced by the expressionof differentcombinationsof a, /? Sieghart, 1989; Doble and Martin, 1992). Using the and y subunits(Sigel et al., 1990; Verdoorn et al., 1990). Xenopus laevis oocyte as a heterologous expression system, the aim of the present study was to examine *To whom correspondence should be addressed. Tel: +44 whether the inhibitory action of the classical GABAA (0)171 753 5897; Fax: +44 (0)171 753 5902; e-mail: receptorantagonists,bicucullineand picrotoxin,could be [email protected]]. similarlydependenton the receptor subunitcomposition. 1289 1290 B. J. Krishek et al.

METHODS antagonist producing 50Y0 inhibition of the agonist response. Cell preparation: Xenopus laevis oocytes The antagonist affinity constants (K~) for bicuculline Xenopus laevis were anaesthetizedby immersion in a were determined according to the method of Arunlak- 0.2% w/v solution of ethyl-m-aminobenzoate(Tricaine). shana and Schild (1959). Schild plots were constructed Stage IV–VI Xenopus oocytes were surgically isolated from measuring dose-ratios obtained from the displace- and microinjected with murine cDNAs in equal ratios, mentsin the GABA concentration–responsecurves in the correspondingto ml, fll, y2S and y2L GABAAreceptor presenceof increasingconcentrationsof bicuculline.The subunits. The cells were cultured in a modified Barth’s best fit line to the data was determinedby the method of medium(MBM) containing(mM): 110NaCl; 1 KC1; 2.4 least squares using unweighed data. In all analyses, the NaHCO~;7.5 Tris–HCl;0.33 Ca(N03)2;0.41 CaC12;0.82 slopes of the unconstrained lines were not significantly MgS04; gentamycin 50pg/ml; pH 7.6 as previously differentfrom unity (P c 0.05, two-tailedt-test).The data described (Krishek et al., 1994). All the experiments were plotted with unconstrained slopes, but since the were performed at room temperature (18–23°C). Cells Schild equation presumes that the antagonist acts in a were used 2 days after incubation and possessed competitive manner, the slopes were also constrained to membrane potentials of —30 to —60mV and input unity, such that pA2= pKB.The individualp~B determi- resistancesof 1–5 MQ. nations were pooled to calculate the mean f SEM. Drugs, solutions and vector construction Electrophysiology: intracellular recording All drugs and solutions were bath-applied. y-Amino- Membrane currents were recorded from Xenopus butyric acid, (–)-bicuculline methobromide and picro- Zaevis oocytes, retaining their follicular cell envelope, toxin were dissolved in water and freshly diluted with using a two-electrode voltage clamp method. Oocytes amphibian Ringer to achieve the appropriate final bath were superfused with an amphibian Ringer containing concentrationsprior to use. (mM): 110 NaCl; 2 KC1;5 HEPES; 1.8 CaC12;pH 7.4. The murine al, fll, y2S and y2L subunit cDNAs were The voltage and current microelectrodeswere filledwith incorporated into the mammalian expression vector, 3 M KC1and 0.6 M K2S04, respectively,and possessed pGWl (British Biotechnology),as described previously resistancesof 5–10 MQ and 1–2 Mf2.An Axoclamp 2A (Krishek et al., 1994). amplifierwas used in voltage clamp mode, and all data were recorded on a Gould 2200 ink-jet pen recorder. RESULTS Analysis of GABA-activated membrane conductance To ascertain the functional roles of the individual The GABA-inducedmembraneconductance(AG) was GABA~ receptor subunits, the inhibitory action of obtained by the subtraction of the resting membrane bicuculline on GABA-induced responses was investi- conductance and subsequentlynormalized (AGN) to the gated using receptors consisting of ulfll, cYl~ly2Sand response induced by 10 ~M GABA. Membrane con- alflly2L subunits. The whole-cell Cl– current and ductance were determined by applying brief hyper- conductance activated by bath-applied GABA (10 vM) polarizingvoltagecommands(–10 mV amplitude,1 sec was inhibited by bicuculline (5–50 PM) in a concentra- duration, 0.2 Hz frequency) to the holding potential of tion-dependentmanner [Fig. l(A)]. –40 mV in the absence or presence of GABA. These Equilibrium concentration–response curves con- data were used for the construction of equilibrium structed for responses to GABA on al~l, al~ly2S and concentration–response relationships for GABA. The alj’ly2L receptor constructsrevealed rightward shifts in data were fitted with an equation of the form: AG/ a parallel manner in the presence of increasing concen- AG~,X= 1/[1 + (EC50/[A])”], where AG and AG~.X trations of bicuculline. The concentration–response represent the GABA-induced conductance at a given curves did not exhibit any depression in the maximum concentrationand the maximumconductanceinducedby response, which is compatible with a competitive mode a saturating concentrationof GABA, respectively.EC50 of antagonism [Figs l(B), 2(A) and 3(A)]. For each definesthe concentrationof GABA ([A]) which induces receptor construct, there are significantincreases in the 50% of the maximum response and n is the Hill EC50values and occasionally in the Hill coefficientsfor coefficient. GABA, measured in the presence of bicuculline (Table The antagonism induced by picrotoxin was analysed 1). by constructing concentration–inhibitionrelationships. From the GABA concentration-responserelationships The data were fitted with an antagonistinhibitionmodel for each receptor construct, the concentration of GABA of the form: AG’/AG = [1– [B/B + IC50]], where AG’ inducing approximately 50?Z0of the maximal conduc- and AG, represent the agonist-inducedconductance at a tance was ascertainedfor the control curve, and also for given concentration in the presence and absence of the curves in the presence of bicuculline. These con- antagonist, respectively. B represents the antagonist centrations of GABA, in the absence and presence of concentration and IC50 defines the concentration of bicuculline,were used to calculate the dose ratio (DR) at Bicuculline and picrotoxin at GABA~ receptors 1291

5PMBicuculline 10KM Bicuculline 50KM Bicuculline

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00 0.0-7 1 -7 -6 -5 -4 -3 -6 -5 -4 -3 Log IB(cucull,ne] Log IBicucullme]

Fig. 1.Antagonismof the GABA-inducedconductancechangeby bicuculline in murine alf?l GABA~receptors. (A) Membranecurrentsand conductancechangesevokedby 10PM GABA(durationindicated by the bar) in the absence and presence of 5, 10 and 50 pM bicuculline at a holdingpotential of –40 mV. (B) NormalizedGABA concentration conductance curves constructed in the absence (0) and presence of 5 KM (.), 50 pM (El) and 100pM (~) bicuculline. The data were fitted to the logistic equation (see Methods; n = 12) and EC50Sand Hill coefficientsdetermined(Table 1).(C) Left: Schildplot of log(doseratio —1)againstlogbicucullineconcentration (M) for the antagonismof the GABA-inducedresponseby bicuculline.The fittedsolid line is determinedby least squaresregressionforunweighed data (withoutslopeconstraint)andthe brokenline indicatesthe %~o confidence limits of the calculatedregression.Right:the same Schildanalysisis presentedwith the regressionline constrained to unity (see Methods).Calculated data from the regressionlines are given in Table 2. each bicucullineconcentrationand for the constructionof firstly,with least square regressionlines fitted to the data the Schildplotsfor the three differentreceptorconstructs withoutslopeconstraintsand with 95% confidencelimits [see Methods; Figs l(C), 2(B) and 3(B); Arunlakshana (derived pK~ values with 95% confidencelimits and the and Schild, 1959]. These plots are shown in two forms: slope of each generated line are given in Table 2); 1292 B. J. Krishek et al. cdply2S Mouse I A 2.5 0 Control ● 5pM Bicuculline ❑ IOpM Bicucdline 2.O m 25pM Bicuculline A 50pM Bicuculline L 100PM Bicuculline 1.5 IL AGN 1.0

/ 0.5

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h 1 00 1 0.0 -7 -6 -5 -4 -3 -7 -6 -5 -4 -3 Log IBlcucullme] Log IBlcucullme]

Fig. 2. Antagonism of the GABA-inducedconductance change by bicuculline in murine alflly2S GABA~ receptors.(A) GABAconcentrationconductancecurvesconstructedin the absence(0) and presenceof 5pM (.), IOpM(0), 25pM (~), 50pM (A) and IOOpM(A) bicuculline. The data were fitted to the logistic equation (see Methods; n = 20) and EC5{)Sand Hill coefficients determined (Table 1). (B) Left: Schild plot with fitted line determinedby least squares regressionfor unweighed data (without slope constraint) and with 95% confidence limits of the calculatedregression.Right:the same Schildanalysisis presentedwith the regressionline constrained to unity (see Methods).Calculated data from the regressionlines are given in Table 2.

secondly,Schild plots are also illustratedindicatingleast toxin of 0.55 t 0.03 pM [Fig. 4(A)]. The mechanism of square regressionlines for the same data with the slopes the inhibition by picrotoxin was studied using equili- constrained to unity, yielding pK~ values for ul~l, brium GABA concentration–response curves. In the alflly2S and ulflly2L receptor constructs (Table 2; presenceof 1 #M picrotoxin,there was a slightrightward Jenkinson, 1991; see Methods). shift in the curve in addition to a depression of the To compare with the inhibitioncaused by bicuculline, maximum response [Fig. 4(A)]. Higher picrotoxin the antagonism of GABA-activated responses by the concentrations (e.g. 10 pM) gave virtually a complete traditional GABA~ receptor antagonist,picrotoxin,was suppressionof the GABA concentration–responsecurve assessed using the GABAA receptor subunit combina- [Fig.4(A)]. The lateral shiftof the controlcurve, coupled tions, al~l, LYl~ly2Sand alflly2L. For oocytes expres- with a decrease in the maximal response, suggested a sing ulfll GABAA receptors, the picrotoxin inhibition complex mode of antagonism for picrotoxin and is curve, constructedfor the antagonismof the response to consistent with a ‘mixed inhibitory’ mechanism (Smart 10 VMGABA, was used to determine an IC50for picro- and Constanti, 1986). The GABA EC50S and Hill Bicuculline and picrotoxin at GABAAreceptors 1293

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— 0.0 A/ 1 I I -7 L-6 -5 -4 -3 -1 -6 -5 -4 -3 Log IBlcucullme] Log IBicucullhnel

Fig. 3.Antagonismof the GABA-inducedconductancechangeby bicuculline in murine w1/31y2LGABA~receptors.(A) GABA concentration conductance curves constructed in the absence (0) and presence of 1 KM (.), 10pM (U) and 50 pM (E) bicuculline. The data were fitted to the logistic equation (see Methods; n = 14) and EC50Sand Hill coefficientsdetermined (see Table 1).(B) Left: Schildplotwith fittedline determinedby least squaresregressionforunweighed data (withoutslopeconstraint) and with 95% confidencelimits of the calculated regression.Right: the same Schild analysis is presentedwith the regressionline constrainedto unity (see Methods).Calculated data from the regressionlines are given in Table 2. coefficients increased in the presence of picrotoxin with a mixed/non-competitive inhibitory mechanism. (Table 3). The GABA EC50S and Hill coefficients were also To determine whether the y2 subunit affected the increased in the presence of picrotoxin (Table 3). antagonism of the GABA-induced responses by picrotoxin, oocytes were injected with ulfily2S and ul~ly2L GABA~ receptor cDNAs. The picrotoxin inhibition DISCUSSIONANDCONCLUSIONS curves for ulflly2S and Lxl/31y2Lconstructs provided The traditional GABA~ receptor antagonists,bicucul- IC50Sof 0.64 ~ 0.1 VMand 0.46 i 0.1 #M, respectively line and picrotoxin, were used to characterize the [Fig. 4(B, C)]. GABA concentration-response curve pharmacologicalproperties of GABAAreceptors and to analyses for each receptor construct revealed that, in determine whether individualsubunits could selectively the presence of picrotoxin (2.5–5 PM), there was also a influencethe potency and antagonistprofileson GABAA small lateral shift in the curve with a concomitant receptorscontainingmurineal~l, cYl~ly2Sand ctl/31y2L depression in the maximum response [Fig. 4(B, C)]. As subunits. For each receptor construct, both bicuculline for ulfll receptors,this mode of antagonismis consistent and picrotoxin were found to be potent antagonists. In 1294 B. J. Krishek et al.

Table1.Analysisof theequilibriumconcentration–responsecurvesforGABA in the absence and presence of bicuculline in Xerropuslaevisoocytes injected with GABA~receptorcDNAs EC5,)(PM) Hill coefficient Subunitcomposition Control + Bicuculline Control + Bicuculline Ulpl 5.87 k 0.7 (5)* 26.9 ~ 2.2 0.96 ~ 0.09 (5) 1.13 t 0.08 (50)* 174.6 t 8.2 (50)” 1.25 t 0.06 (1OO)*356.3 ~ 20.8 (1OO)*1.29 t 0.09 al~ly2S 10.03 * 0.2 (5)* 52.6 f 2.8 1.3 f 0.03 (5) 1.43 * 0.1 (lo)* 100 * 3.5 (lo)* 1.5 ~ 0.07 (25)* 156.4 t 8.1 (25)* 1.6 f 0.09 (50)’ 256.7 + 21.8 (50)’ 1.55 ~ 0.06 (1OO)*554.1 f 16.4 (loo)* 1.5 * 0.07 ctlflly2L 10.20 f 0.5 (l)* 19.9 * 0.97 1.22 f 0.06 (1) 1.2 * 0.05 (10)* 66.4 ~ 4.1 (lo) 1.3 * 0.09 (50)” 319.6 t 38.0 (50) 1.2 i 0.1 EC5,)and Hill coefficientdata were determinedin the absence and presenceof bicucullinefrom fittingthe logistic model to the normalized curves (see Methods),with all values being the mean~ SEM. Numbers in parentheses indicate the bicuculline concentration(PM); *Significantchange from control (P<0.05).

Table 2. Analysis of bicucullineantagonismof the GABA-inducedresponse in recombinantGABA~ receptors Subunit pKB pK~ composition Unconstrainedslope (unconstrainedslope) (constrained slope) alfll Mean 0.92 pKB 6.00 5.87 Upper C.L. 1.08 Upper C.L. 6.25 Lower C.L. 0.76 Lower C.L. 5.75 al/ll;,2S Mean 0.88 pK~ 5.97 5.83 Upper C.L. 1.04 Upper C.L. 6.24 Lower C.L. 0.72 Lower C.L. 5.69 ctl/lly2L Mean 0.87 pK~ 5.99 5.86 Upper C.L. 1.08 Upper C.L. 6.22 Lower C.L. 0.67 Lower C.L. 5.77 Values are derived from the Schild plots of pooled data shown in Figs 1, 2 and 3. 95% confidence limits (C.L.) of the calculated regressions were determined for lines generated without slope constraint. The data were also subjected to linear regression analysis and the slope constrained to unity. agreementwith previousstudies,bicucullineappeared to tional limitationscould arise from the prospect of uptake act as a competitiveinhibitor,shiftingthe dose–response of the agonist, or insufficient equilibration of the curve to the right with no depression in the maximum antagonist.Unlike brain slice preparations, the Xenqms response(cf. Simmonds,1982).The apparentpKBvalues oocyte expression system apparently does not contain determinedin this study suggestedthat the affinityof the any GABA uptake sites, which should result in a more antagonist appears to be relatively unaffected by the accurate determination of pKB.Moreover, equilibration subunit isoforms present within the GABAA receptor of the antagonistwas checked by repeated administration construct. of the same concentration of GABA to ascertain that a Schild analysis of the dose–response curves in the steady-state inhibition was achieved in the presence ‘of presence of bicucullinegave a slope for the Schild plots bicuculline (and picrotoxin) prior to building each for each receptorconstructof approximately1, indicating concentration–responsecurve. that bicuculline could be acting as a competitive It is interesting to compare the pKB for bicuculline antagonistat these recombinantGABAAreceptors.Even determined in this study with the values reported from though bicuculline may be a classical competitive neuronalpreparations.Using the rat cuneate nucleusslice antagonist, there are various factors that could distort and extracellular field potentials, a pKB value for the theoretical predictionsof a unitary Schild plot slope bicuculline of 5.93 has been reported (Simmonds, that will affect the estimationof the affinityconstant.The 1982). Furthermore, bicuculline antagonizedthe isogu- Hill coefficients for GABA determined for all of the vacine and muscimol-inducedinhibitionof hippocampal subunitcombinationsutilized in this study approximated populationspikes with apparent pKBvalues of 6.24 and to 1. It is debatableas to whether these values accurately 6.10,respectively(Kemp et al., 1986),and for embryonic reflect the underlying stoichiometry of the agonist– chick whole-brain, mRNA-injectedXenopus oocytes, a receptor interaction; however, for the present analysis, pA2of 5.93 has been reportedfor bicucullineinhibitionof the assumption of an n“ of 1 was used to simplify the GABA-activated responses (Smart et al., 1986). The resulting Schild analysis (cf. Simmonds, 1990). Addi- similarity of the pK@ for bicuculline determined for Bicuculline and picrotoxin at GABAAreceptors 1295

alpl Mouse

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———..—- al ply2L Mouse:’ c ‘“”pzzia /- 1.5-

AGN 1.O-

0.5-

0 0.1 1 to 100 1000 Picrotoxin Concentration (pM) GABA concentration (PM) Fig. 4. Inhibition of the GABA-inducedconductance change by picrotoxin on oocytes expressing murine alfll, ulflly2S and Ixl/?ly2LGABA~receptors.(A)-(C) Left column:picrotoxininhibitionplots were constructedfor the responseto 10pM GABA recorded on receptors composed of al/?l (A), ctl/lly2S (B) and al#ly2L (C) subunits. The picrotoxin inhibition curves are normalized with respect to OYOinhibition in the presence of 0.05pM picrotoxin (defined as 100% control GABA-activated conductance).All data were fitted accordingto the antagonistinhibitionmodel (see Methods;n = 9) and IC50values determined. Right column: for each receptor construct, GABA concentrationconductance curves were constructed in the absence (0) and presence of 1PM (.) and 10pM (Q; alfll) picrotoxin (A); 5 flM (.; czlflly2S)picrotoxin (B); and 2.5 pM (.; ul/31y2L) picrotoxin (C). The data were fitted to the logistic equation (see Methods; n = 18) and EC50Sand Hill coefficients determined (Table 3). 1296 B. J. Krishek et al.

Table 3. Analysis of picrotoxinantagonismof the GABA-inducedresponse in recombinant GABA~ receptors EC50 Hillcoefficient Subunit .— composition Control + Picrotoxin Control + Picrotoxin cil/11 5.87 t 0.7 (l)* 7.5 i 0.45 0.95 + 0.09 (l)* 1.37 i 0.1 ctlp172s 10.03 i ().2 (5)” 13.5 t 0.8 1.3 f 0.03 (5)* 1.5 f 0.1 c!lflly2L 10.2 f 0.5 (2.5)* 31.2 t 1.6 1.22 ~ 0.06 (2.5)* 1.5 ~ ().1 Analysisof the equilibriumconcentration–responsecurves for GABAwas completedin the absence and presence of picrotoxin. EC5(,and Hill coefficientdata were determinedfrom fittingthe logistic model to the normalizedcurves from Fig. 4 (see Methods), with all values being the mean ~ SEM. Numbers in parentheses indicate the picrotoxinconcentration(pM); *Significantchange from control (P<0.05). neuronalor expressedGABAAreceptors,usingresponses hinderingion channelactivation,thereby initiatinga non- derived from multicellular preparations or mRNA- competitive depression in the dose–response curve injected Xenopus oocytes, and therefore likely to (Smart and Constanti, 1986). However, this type of represent a heterogeneouspopulation of GABAA recep- mixed inhibitioncould also arise from picrotoxinbinding tors, indicatesthat the antagonismby bicucullinein these to a singlesite that is distinctfrom the agonistrecognition studies is also probably independent of the subunit site and also from the ion channel (Smart and Constanti, composition.The similarityof the pKBSdeterminedfrom 1986). the present study using recombinant GABA~ receptors Similar to bicuculline,the picrotoxin inhibitioncurves with the neuronal pKBdeterminationsalso supportsthis and the corresponding IC50 values suggested that the conclusion. potency of this antagonist appears to be relatively There are many differentpermutationsof subunitsthat unaffected by the subunit isoforms present within the can be expressed to form functional GABAAreceptors. GABAAreceptor. In comparison, an IC50of 0.6PM has Presently, the expression of bovine alflly2L GABAA been reported for picrotoxin antagonism of GABA- receptors revealed a similar pKBvalue for bicucullineof activated responses using chick brain mRNA-injected 6.4 (Home et al., 1992);however, an estimatedpA2of 5, Xenopus oocytes (Sigel and Baur, 1988), and for rat derived from two concentrations of antagonist, was recombinantal fll;’2, u5/32y2,xl f12y2and f12y2GABAA reported for the bicuculline inhibition of responses to receptors, IC50S of approximately 1 pM have been GABA on human a6/?3y2S receptor constructs. This determined (Sigel et al., 1990). Interestingly, whilst /?1 would be compatible with a potential reduction in the subunit homomers (and also ~3 homomers, J. W. potency of bicuculline on these recombinant receptors Wooltorton and T. G. Smart, unpublished observations) (Thompson et al., 1996). Coexpression of the insect are sufficientto formpicrotoxinbindingsites(Blair et al., (Drosophila) GABA receptor subunits, resistance to 1988; Sigel et al., 1989; Krishek et al., 1996; IC50of dieldrin (Roll)and a homologue of the vertebrate GABAA 2.1 #M), additional binding sites may be provided for receptor ~ subunit in Sf21 cells, produced receptors picrotoxin by other subunit combinations, since u3u5y2 displaying differential sensitivities to picrotoxin and bicuculline (Zhang et al., 1995a). Rdl homomers were GABAA receptors also exhibit a sensitivity towards qualitativelycategorized as “sensitive to picrotoxin and picrotoxin (Sigel et al., 1990). The relative insensitivity insensitiveto bicuculline”, whereas the potentialhetero- of picrotoxin to the subunit compositionof the GABAA mers (RdZ + P homologue) were suggested to be receptor is possibly not surprising given the recent “sensitive to bicucullineand insensitiveto picrotoxin”. mutagenesis studies in insect and mammalian GABA In the present study, the inhibition of the GABA receptors,indicatinglikely amino acid residuesimportant concentration–responsecurveby picrotoxinfor the ulfll, for antagonist binding. For the Rdl subunit, an alanine ul~ly2S and ulflly2L constructswas of the mixed/non- (A) to serine (S) mutation at position 302 in the second competitive type, i.e. shifting the dose–responsecurve transmembrane domain (TM2) renders the GABA- with a reduced maximum response. The nature of the activated membrane current insensitive to picrotoxinin inhibitionby picrotoxin was fully reversible, even after (ffrench-Constant et al., 1993). In comparison, for quitehigh concentrations(100 ,uM),suggestingthat there mammalian GABAAreceptors, mutation of the aligned was little or no slowly reversible/irreversiblecomponent threonine (T) residue at positions 261 (rat al), 246 (f12) to the inhibition. An irreversible component would be and 271 (y2), rendered the heteromeric al f12y2receptor manifest by a clear non-competitivedepression in the insensitiveto picrotoxin(Gurley et al., 1995).Moreover, GABA concentration–responsecurve. Another possible co-expression of the mutant f12 (T246F) subunit with mechanismof inhibitionfor picrotoxincould involvetwo wild-type al and y2 subunits was sufficient to confer distinct binding sites present on the GABAA receptor, upon the receptor an insensitivity to picrotoxin. These providing a competitive inhibition of GABA binding at threonine residues are highly conserved among GABAA the agonist recognition site, and also by allosterically receptor subunits, and thus may account for the Bicuculline and picrotoxin at GABA~ receptors 1297 independenceof picrotoxinantagonismfrom the subunit Gurley D., Amin J., Ross P. C., Weiss D. S. and White G. compositionof the GABAAreceptor. (1995) Point mutations in the M2 region of the a, /3 or y Apart from the conservedthreonineresidue, alignment subunit of the GABAA channel that abolish block by of the TM2 region of the al mammalian GABA~ picrotoxin.Receptors and Channels3: 13-20. receptor subunit with the corresponding region in the Home A. L., HadinghamK. L., MacauleyA. J., Whiting P. and Rdl subunit, and also by replacing selected residues in Kemp J. A. (1992) The pharmacology of recombinant GABAAreceptors containing bovine al, ~1, y2L sub-units TM2 with cysteines (substituted-cysteine-accessibility stably transected into mouse fibroblast L-cells. Br. J. method), has implicated a valine (V257) residue in the Pharmac. 107: 732–737. binding site for picrotoxin (Xu et al., 1995).This valine JenkinsonD. (1991)How we describecompetitiveantagonists: occupies a correspondingposition to the alanine (A302) three questionsof usage. TrendsPharmac. Sci. 12: 53–54. residue in Rdl that is importantfor picrotoxinsensitivity; Kemp J. A., Marshall G. R. and Woodruff G. N. (1986) however, the valine is not conserved in the mammalian Quantitativeevaluationof the potenciesof GABA-receptor /11 and 72 subunits. A similar alignment of the TM2 agonistsand antagonistsusing the rat hippocampalslice regions for Rdl and the human pl and p2 subunits, preparation.Br. J. Pharmac. 87: 677-684. suggeststhat A302 also correspondsto a proline (309) in KrishekB. 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