INVOLVEMENT OF HISTIDINE 134 IN THE BINDING OF α-BUNGAROTOXIN TO THE NICOTINIC ACETYLCHOLINE RECEPTOR

Pergamon

@

S01974186(96)00069

Neurochens. Int,Vol.31,No.1,pp. 151–157, 1997 ~ 1997ElsevierScienceLtd Printedin GreatBritain.Allrightsreserved 0197-0186/97 $17.00+0.00

INVOLVEMENT OF HISTIDINE 134 IN THE BINDING OF ct-BUNGAROTOXIN TO THE NICOTINIC ACETYLCHOLINE RECEPTOR GRACIELA D. VENERA, FERNANDO D. TESTAI, CLARA PEIiA, HUGO D. LACORAZZA* and MIRTHA J. BISCOGLIO DE JIMENEZ BONINOI Instituto de Quhnicay FisicoquhnicaBiological(UBA-CONICET),Facultad de Farrnacia y Bioquhnica,Junin 956(1113),BuenosAires, Argentina (Received 14 March 1996; accepted 13 June 1996)

Abstract-Peptides correspondingto the sequenceu 124-147of the Torpedo cah~ornicaand Homo sapiens nicotiniccholinergicreceptors were synthesized.The His residue at position 134was ethoxyfortnylatedor substitutedbyAla. Effectsof suchmodificationswerestudiedby:(a)a toxinblot assayand (b) a competition ~.bungarotoxin,in solution. assay between each peptide and the Discopyge tschudii receptorfor 12SI Apparent Kd values were 0.1 and 0.8PM for Torpedo calijornica and Homo sapiens nativepeptides, respectively, andno bindingwasobservedwhenthe Hisresiduewasmodifiedor substitutedby Ala. ics~ values forthe Torpedo calt~ornicaand Homo sapiens fragmentswere 1.0and 0.8PM, respectively,and no significantdisplacementoccurred when His 134was ethoxyformylatedor substituted by Ala. Hydroxylamine treatment restored 8W100°/0of their binding ability. Results strongly support the involvementof His 134in the binding of a-bungarotoxineither to the Torpedo call~ornicaor the Homo sapiens receptor. ~ 1997 E1sevierScienceLtd

Tronconi et al., 1990;Donnelly-Roberts and Lentz, 1991;McCormick et al., 1993)and fusion proteins containing receptor sequences (Barkas et al., 1987; Gershoni, 1987)—leadto the conclusion that there is a major ct-BgTxbinding site betweenresidues 173and 204 of the a-subunit. Additionally, different results have been reported for the cys-loop, an even more conserved region flankedby cysteineresiduesat positionsa 128and 142. While severalgroups (Gotti et al., 1988;Criado et al., 1986;Griesmann et al., 1990;Neumann et aL, 1986) concludethat the regionis not involvedin the binding sites of the receptor, other researchershave proposed it as a major determinant of cholinergicand/or neurotoxin binding sites (Cahill and Schmidt, 1984; McCormickand Atassi, 1984;Smart et al., 1984;Wilson et al., 1985;Mishina et al., 1985;Mulac-Jericevic and Atassi, 1986,1987,1988;Luyten, 1986;Cockroft et al., 1990;Schroeder et al., 1994;Fulachier et al., 1994;Klukas et al., 1995). On the other hand, previouswork in our laboratory *Presentaddress:NationalInstitutesof Health,National was done in order to contribute to the understanding Eye Institute. Geneticsand MolecularImmunology Section,Bldg 10 Room ION116Bethesda, MD 20892, of the structur~function relationships of the LGIC family. We purified and characterized the receptor U.S.A. from Discopyge tschudiielectric organs,whichis structAuthor to whom all correspondenceshouldbe addressed.

Ligand-gatedion channels(LGIC) constitute a superfamily of membrane proteins involvedin the nervous system signal transduction whose best characterized member is the nicotinic acetylcholine receptor (nAChR). The nAChR from fish electric organ and skeletal muscle (mol. wt about 300000) is a heterologous transmembrane pentamer assembled with a stoichiometry u2~@. Different approaches have been used in order to localize the ACh and a-bungarotoxin (c+BgTx)binding sites. a-BgTx binds to the receptor with high affinity and competitively blocks the depolarizing effectof ACh. Results of these studies—u-BgTxbinding to isolated subunits (Haggerty and Froehner, 1981;Gershoni et al., 1983),proteolytic peptide fragments (Tzartos and Changeux, 1983;Wilson et al., 1984;Pedersenet al., 1986),syntheticpeptides(Wilson et al., 1985, 1988;Mulac-Jericevicand Atassi, 1986; Neumann et al., 1986; Ralston et al., 1987; Conti-

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turally and biochemicallyvery similar to that from Torpedo calzfornica (Ochoa et al., 1983).We have also studied the effectof specifichistidine modificationof the receptor with ethoxyformic anhydride. Ethoxyformylation of the native-membranereceptor as well as that purifiedby affinitychromatography induces a loss of ct-BgTxbinding capacity, time and reagentconcentration dependent, and restored by ethoxyformylation reversion. Moreover, consistent with the modificationof a binding site, the ethoxyforrnylation does not bear on the affinitybut reduces the number of receptors. Additionally, in the presence of carbamylcholine,ethoxyformylationshows some ligand protectiveeffects.Those results indicate an important role for histidine residuesin the receptor-toxin interaction (Lacorazza et al., 1992Lacorazza et al., 1995). Therefore, we were interested in examiningthe possible role of His 134located at position 7 of the cysloop in the toxin binding to the u-subunit of Torpedo calz~ornica and Homo sapiens receptors. For this purpose, peptides u 124-147of the sequenceof the Torpedo call~ornica and Homo sapiens M-subunitswere synthesized and the ethoxyforrnylatedpeptides and those including Ala instead of His at position 134, were used. EXPERIMENTAL PROCEDURES Materials

column (from AtIi-Gel 10 and bromoacetylcholine),previously equilibrated with Buffer A containing 10/. sodium cholate and washed with 4 column vols of Buffer A containing 17. sodiumcholate and 2mg/ml lecithin;the nAChR waseluted with 50ml of IOmMcarbamylcholinein the same bufferand the ehratewasimmediatelydialysedagainst Buffer A and stored at –70”C. Peptide synthesis

Peptide synthesis was performed by the classical solid phase method of Merrifield (1963)and cysteine oxidation by using thalium trifluoroacetate. Peptides a 12*147 were synthesizedas per the sequence124147 of the Torpedo californica (Tc.i,: F K S Y C E I I V T H F P F D Q Q N C T M K L G) and Homo sapiens (Hs~i,:F K S Y C E I I V T H F P F D E Q N C S M K L G) receptor a-subunits, respectively. Peptides u 124147 (Tc~,.) and u 12+147 (Hs~l.) were synthesizedwith Ala instead of His at position 134.Cysteine oxidation was achievedby the method of Lam-Thanh et al. (1989). Crude materials were purified to homogeneityby reverse phaseHPLC on aVydac C18column(25cm x 4.6mm) using an 8+00/. acetonitrile gradient in O.1°/0trifluoroaceticacid, at a flowrate of 1ml/min. The sequenceof each peptide was determined. Amino acid analyses

Sampleswerehydrolyzedin vacuum-sealedtubes at 1IO°C for 20h inconstant boilingHClcontainingphenol (1mg/mI). The hydrolysateswereanalysedin a Beckman119CL amino acid analyser, Methionine and cysteinewere determined as methionine sulfone and cysteic acid respectivelyafter perforrnicacid oxidation.

Discopyge tschudii and Torpedo calfornica electric organs Peptide sequencing werekindlyprovidedby Dr Barrantes(INIBIBB,Bahia Blanca,Argentina)andBaxterR&DEurope,S.C.(Nivelles, It was performed in the Facility for Peptide and Protein Belgium),respectively, andwerestoredinliquidnitrogen. Sequencing (LANAIS-PRO,Universityof BuenosAires).An a-BgTxfrom Bungarus muhicinctus, ethoxyformicanhy- Applied Biosystemsgas-phase automated sequencer model

dride, L-a-phosphatidylcholine(L-a-lecithin)from soybean, and carbamylcholinewere obtained from Sigma Chemical Co., St Louis, MO, U.S.A. AtIi-Gel 10 was from Bio-Rad (Richmond, CA, U.S.A.), and the carrier-free ‘251Nafrom NEN ResearchProducts, Du Pent Company(MA, U.S.A.). Bromoacetylcholinewas synthesizedby reaction of choline bromide with bromo-acetylbromide from SigmaChemical Co. and Aldrich Chemical Co., Inc. (WI, U.S.A.), respectively.N-t-Boc glycinechloromethylresin ester, N-t-Boc-Lamino acids, trifluoroacetic acid, triethylamine and dicyclohexylcarbodiimidewerepurchasedfrom SigmaChemical Co. (St Louis,MO, U.S.A.).All other reagentsused wereA. R. grade.

470Awas used. Degradation steps were carried out by using the 03RPTH program. Following each cycle, the phenylthiohydantoinamino acid was identifiedby using an on-line reverse-phaseHPLC system.

Histidine modl~cation

Tc~,, and Hs~,, peptide solutions (0.5–250#g/100pl)in IOmM MOPS buffer, pH 7.4, IOOmMNaCl, 0.27. Triton X-1OO(NMT,W),were modifiedduring 30min at room temperature by ethoxyfonnic anhydride (2mM final concentration). For kinetic studies the ethoxyforrnylation reaction was carried out in the cellof the spectrophotometer at 25”C. Ethoxyforrnic anhydride (10 pl) in ethanol was added to 1ml of the NMT,Wpeptide solution (2mM final Extraction andpurlJication of the receptor concentration). The absorbance increase at 242nm was recThe method of Ochoa et aL (1983)was usedfor extraction orded until a plateau was reached. The ethoxyfonnylation and purification, modified as follows:membranes (protein extent was calculated by using a molar extinctioncoefficient concentration: l&20 mg/ml)were diluted to 2.3mg/ml with of ethoxyfonnylimidazolin proteins of 3.9x 103M-’ cmIOmMphosphatebuffer, IOOmMNaCl, 0.1mM EDTA and at 242nm (Choonget al., 1977);data analysiswasperformed 0.02Y0(w/v) NaN3, pH 7.4 (Buffer A) supplementedwith by utilizing a non-linear curve fitting program. De-ethoxysodium cholate to a final detergent concentration of 10/0 forrnylationwas achievedin 1h by adding a hydroxylamine (w/v). After centrifugation at 66,000g, 45 rein) the super- solution(O.2Mfinalconcentration)to theethoxyfonnyl(EF) natant (AzgOn~ = 1.5)was applieddirectlyonto a 25ml affinity peptide solutions.

ct-Bungarotoxinbindingto the nicotinicacetylcholinereceptor Circulardichroism measurements CD measurements were performed in the 203-250 nm range at 25°C in a Jasco J 20 spectropolanmeter. Protein concentration was 0.05mg/ml in Buffer A containing 1°/. sodiumcholate, a-BgTxradiolabelling

The ct-BgTxiodinationwas performedaccordingtoLacorazzaeral (1992).a-BgTx(10 nmol) was dissolvedin50pl of 0.1M phosphate buffer, pH 7.4, to which 0.5mCi of carrier-free ‘2’INa was added, followed immediately by 5nmol of Chloramine-T. The reaction was allowed to developfor 2min at room temperature and was stopped by addition of 6nmoIof sodiummetabisulfitein 2PIof distilled water, and by dilution with 70pl of the same phosphate buffer.The reaction mixture was chromatographedthrough a SephadexG25column(62x 0.7cm)equilibratedand eluted with 0.1M phosphate buffer, pH 7.4. The fractions containing the ‘2’Ia-BgTxwere pooled and stored at –20°C. The specific activity was 1(L5OCi/mmol. Toxin concentration was calculated according to Lindstrom et al. (1981). Toxin blot assays

Aliquots (1-51d)of peptide solutions(1mg/ml)in IOmM sodiumphosphate bufferpH 7.4, 140mM NaCl (PBS)were spotted onto nitrocellulosestrips and allowedto dry. Nonspecificbindingwas reducedby preincubationwith a ‘blocking solution’(5mg/ml of cytochromeC and albumin 10/0in PBS)for 2hat room temperature.The blockednitrocellulose strips were incubated overnight at 4°C with 0.014.15PM IM1u-BgTx (3_6~ IOGcpm)in PBSwith 5mg/mlcytochrome C and 0.1% albumin. The toxin blots were washed eight times with 2ml of PBS containingO.lO/.Triton X-1OOfor 3min each. Radioactivity of the blots was measured in a gamma counter. The non-specificbinding of ‘251cr-BgTx at each concentration of ‘251a-BgTx was determined by preincubation of the blocked strips with utdabelled cr-BgTx (100PM) in blockingsolution 2h at room temperature. All assayswere performedin triplicate. Relative peptides affinity was compared by calculating an apparent Kd using the EBDA and LIGAND programs (Munson and Rodbard, 1980;McPherson, 1983).

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with buffer.The 100Yobindingwas estimated by incubating the receptor with radiolabelled toxin. The IC50values were determined using the EBDA and LIGAND computer programs (Munsonand Rodbard, 1980;McPherson, 1983).The results are also presented as a Hofstee plot in whichthe IC50 valuescan be directlycompared as the negativeslopesof the linear regressionlines. Protein concentration

Protein concentration was measured according to Lowry et al. (1951).For kinetic studies protein concentration was

determinedby amino acid analysis.

RESULTS Synthetic peptides

Reversephase HPLC of crude materials yielded,in all cases, a major fraction (65–75°/0of total absorbance). Cysteine oxidation was performed by using thalium trifluoroacetate in order to prevent the formation of undesirable disulfidebridges. All peptides werecharacterizedby microsequencinganalysiswhich showed that their sequences fully agree with those expectedfrom the receptor covalent structure. Toxin blot assays

Peptides(1mg/ml)and receptors (0.09mg/ml) were spotted onto nitrocellulosestrips. After preincubation with the blockingsolution, in the presenceor absence of IOO,aMunlabeled c+BgTx, the strips were incubated with 1251~-BgTx in the blocking solution and dried after washing. Experiments were performed in triplicateand radioactivityof the spots wasthen determined in a y-counter. In three independent experiments, preincubation with unlabeled c+BgTxreduced the total 1251a-BgTx bound to peptides by 70%. The concentration dependenceof ‘251a-BgTx bindCompetitive inhibition of ‘2sIa-BgTx binding by synthetic ing was assessed over a range from 0.01 to 0.15pM peptides IZ51a-BgTxas described in Experimental Procedures. Usingthe method of Schmidtand Raftery (1973),peptides The apparent K~ valueobtained from the Scatchard TcHi,,HsHi,,Tc~l.,Hs~l.,those ethoxyformylatedat His 134 analysisfor Tc~i$a 12*147 was O.I Of 0.03pM (B~.X: and those that werede-ethoxyformylated(0.5–250#g/100#1) were preincubated overnight with 2pmol of ‘2sIa-BgTxin 1.1x 10-’1M). When the His residue of peptide Tc~i~ NMT,OO, pH 7.4,at 4“C.Discopyge tschudiireceptor(1pmol) a 124-147was ethoxyformylated,it lost its toxin bindwas added to 100pl of the peptide-toxin solutionsand after ing capacity. In order to discard any conforrnational 3min of incubation at room temperature the mixtureswere change that could be responsiblefor the lack of toxin filtered through DEAE+ellulose disks previously washed with 10mM MOPSbufferpH 7.4, IOmMNaCl, 0.2°/0Triton binding, peptide CD spectra were determined. The X1OO(NMT,O).The filters were then washed three times spectra of native and modified peptides (not shown) with 3ml of NMTIOand radioactivity was measured. Five were superimposable;besides,a minimum at 216 nm experimentsin duplicate were averaged. Non-specificbind- indicated that they possess the /l-structure expected ing was measured by incubating the receptor with a large on the basis of modelling studies. Therefore, ethoxyexcess of c+BgTxfor IOmin before adding the ‘251u-BgTx. in the presenceof peptides was com- forrnylation did not induce peptide confirmational Bindingof U51a-BgTx pared with values obtained with toxin preincubated only changes and results suggested a direct relationship

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G. D. Venera et al.

between His modification and the loss of toxin binding. To further confirmthesedata, His 134waschemically substituted by Ala;CD spectra showedabsence of confirmational changes and no toxin recognition was observedin this case either. As the His residue in the Tc~i,u 124-147fragment was found to be critical for u-BgTxbinding, and in order to investigate if this is a common feature in receptorsfrom differentorigins,a toxin blot assaywas performedwith segmentHs~i,a 124-147.The peptide bound ‘251ct-BgTx with a slightly lower affinity; an apparent K~ value of 0,80~ 0.17AM (Bmax: 1.6x 10-” M) was obtained. The K~ value for both Torpedo caltfornica and Discopyge tschudii receptors obtained in a parallel experimentwas 3 x 10-9M. Competitive inhibition synthetic peptides

of

‘251u-BgTx

binding

(a) 100 S

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1 I I I I 1 I n -%.0-7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -: ,5

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It is well known that in solid-phasebinding assays the immobilizedpeptides could suffer a loss of their binding activity as a consequenceof adopting a more rigid conformation, which may be unfavorable for binding. Based on these considerations, we also used a competition assay in which the native peptides, the % Displaced/[Peptide, rIM] I I I I I 1 ethoxyformylated ones and those including Ala %.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -: ,5 instead of His at position 134,werepreincubatedwith log [Peptide,M] ‘2%-BgTx and then incubated with the Discopyge tschudii receptor, as detailed in Experimental Pro- Fig. 1.Competitionof Torpedo calfornica and Homo sapiens peptides for ‘z’Ict-BgTx.Peptides (concentration: 0.5– cedures. 250pg/100pl)were incubated overnight at 4°C with 2pmol Figure l(a) shows results of a typical experiment of IZSIu-BgTx prior to the addition of 1pmol of Discopyge (n =4) for native and ethoxyformylatedTc~i~as well tschudii receptorand assessedas describedin Experimental as for Tc~l..The nativepeptidecompetitivelyinhibited Procedures.Theinsertsshowtheconcentrationdependence as HofsteeplOtS.(a) the ‘z51c+BgTx bindingto the Discopyge tschudiirecep- of peptidecompetitionfor lqSIu.BgTx ● , EF-Tc~i,; ■ , Tc~,.. ( b)A, Hs~i,;(), EF-Hs~i,; A, TC~i.; tor, the maximum inhibition obtained under these ❑ , Hs~la. experimentalconditionsbeing60°/0.Tc~l,and ethoxyformylated Tc~i,inhibited the binding only by 5 and 3~o, respectively,thus confirmingresuks from toxin blot assays. Moreover, hydroxylamine treatment, IC50values were 1 and 0.8pM for Tc~i3and Hs~i,, which leads to de-ethoxyformylationof the modified respectively; therefore, their potency as the native peptide, restored 80Y0of the binding ability. Similar results were obtained with the Hs-cor- receptor competitors for ‘251a-BgTxis comparable. responding peptides (Fig. l(b)). The maximum inhi- The insetsin Fig. l(a) and (b) showresults as Hofstee bition reached in this case was 58, 3 and 2Y0for HS”,S, plots. Figure 2 summarizesresults obtained when native, ethoxyformylatedHs~i,and Hs~l. respectively.In all the above experiments,the value for 1251wBgTx bind- ethoxyformylated,and de-ethoxyformylatedpeptides, ing in the absence of peptides was considered to be at a concentration of 70AM, were submitted to the 100VO.On the other hand, inhibition of ‘251cz-BgTxsolution competition assay. Data are representative binding by bovine serum albumin (25pg/100pl) was of five independent experiments, each performed in taken as a negative control (under 5Yo), though the duplicate. De-ethoxyformylation restored 80 and IOOYO of the modifiedTc~i,and Hs~i~binding ability, lack of response of HsA1a(whose structure only differs respectively,and peptidesincludingAla instead of His from that of the native peptide as it has Ala instead at position 134,were almost totally displaced by the of His at position 134) is the best negative internal nAChR. control.

‘or 11-

c+Bungarotoxinbindingto the nicotinicacetylcholinereceptor

Fig. 2. Competition of peptides with the Discopyge tschudii receptor for ‘251a-BgTx.Open bars: Tc~i,, EF-Tc~i,, hydroxylaminetreated EF-TcHi,,and TCA1.; hatched bars: Hs~i,,EF-HsHi,,hydroxylaminetreated EF-Hs~i,,and Hs~,a. Peptides (70pM) were preincubated overnight at 4°C with IZSI ~.BgTx(20~M, 380cpm/fmol)inafinalvolumeof 100P1 Discopyge tschudii ofNMT,Wbuffer,pH7.4.Thesolubilized

nAChR was added (final concentration: IOnM) and, after 3min incubation, the solution was filtered and radioactivity measuredafter washing(further details in ExperimentalProcedures).The valueswerecorrected for non-specificbinding, and 1251 ct-BgTxbindingin the absence of peptides was considered to be 1000/o.Data are representative of five independent experiments, each performed in duplicate. Bars represent standard errors. DISCUSSION The apparent Kd value of Tc~i, (0.1 ~M), obtained from the solid phase assay, indicates that the fragment 124-147 is involved in the binding of the u-BgTx to the receptor in agreement with data obtained by Atassi and co-workers (McCormick and Atassi, 1984; Mulac-Jericevic and Atassi, 1986;Mulac-Jericevic and Atassi, 1987). Hs~i, bound ‘251a-BgTxwith approximately eight times less affinity. This result also agrees with previous findings indicating that synthetic fragments corresponding to the human receptor a subunit have binding properties different from those of the Torpedo receptor segments:peptidesflankingresidues

192 and 193 in the human receptor a subunit have only a weak binding capacity, while peptides corresponding to the Torpedo callfornica u-chain containing such residues show considerable binding ability to u-bungarotoxin and c+cobratoxin (Neumann et al.,’ 1986;Mulac-Jericevicand Atassi, 1986, 1987;Gotti et al., 1988;Ralston et al., 1987). As expected, peptides display 10W1OOO times less affinitythan Torpedo callfornica or Discopyge tschudii receptors,thus supportingthe hypothesisthat the neurotoxin recognizesmultiple sites on the u-subunit. The His residue of fragments a 124-147(Tc~i,and Hs~i,)was either ethoxyformylatedor substituted by Ala; lack of their binding capacity suggeststhat His

155

134 is a key residue for peptide-toxin interaction. Differencesobservedin the binding of a-BgTxto Tc~i, and Hs~i,peptides in toxin blot assays could not be accounted for becauseof differencesin the B~.X values calculated by this method. As regards the competition assay in solution, peptide relativeaffinities(measuredas ic50values)were 1.0 and 0.8PM for Tc~i,and HsHi,,respectively;therefore, their potencies as the native receptor competitors for IZSIa-BgTxare Comparable. Comparison of these values with that obtained for the Torpedo calzfornica al 181–200 fragment (McLane et al., 1991)indicates that the a 126147 fragment is able to compete for the neurotoxin as efficientlyas the former. This agrees with previous works (McCormick and Atassi, 1984; Mulac-Jericevicand Atassi, 1986, 1987;Conti-Tronconi et al., 1990)and with the proposal of Atassi and co-workers that several discontinuous fragments of the a subunit nAChR could bind the toxin, including the segmentcontaining Cys 128and 142(McCormick and Atassi, 1984;Mulac-Jericevicet al., 1988).Further experimental support for this hypothesis comes from works by Conti-Tronconi et al., 1989;Dennis et al., 1988;Fulachier et al., 1994.Additionally,it is also wellknown that the binding surface of u-neurotoxins extends over a large receptor area (Love and Stroud, 1986),even interacting with multiple subunits. On the other hand, peptide de-ethoxyformylation results in the restoration of binding ability (Fig. 2). These data strongly support that His 134,belonging to the cys-loop 128–142,participates in toxin binding either to the Torpedo calz~ornicaor the Homo sapiens receptor, thus confirmingthat the neurotoxin binding site involves amino acids from several loops of the receptor N-terminal hydrophilicdomain. Furthermore, snake muscle nicotinic receptors are resistant to a-BgTx,as is evident by the absence of uBgTx binding to them (Halvorsen and Berg, 1986). The snake receptor sequencesare of great help in the identification of regions that may be involved in the interaction with neurotoxins. Amino acid sequences for residues 119–222of the cobra (Naja naja atra) and water snake (Natrix tesse[lata) IXsubunits have been determined (Neumann et al., 1989).Sequence comparison shows an homology of 78?4.with other u subunits. It is likelythat those amino acids conserved in other receptors but not present in cobra and water snakes may interact with a neurotoxins. All affinitylabelled amino acid residues are included in the sequenceof the snake musclea subunit (Neumann et al., 1989);however, the His residue at position 134, highlyconservedin nAChR, is not presentin the snake musclereceptor.

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Moreover, Cockroft et al. (1990)have modeled the cys-loop subregion. The binding agonist complex model includes: (a) the invariant aspartate 138, located at position 11of the cys-loopand considered to be an anionic site that could interact with the positivelycharged amino group of agonists;(b) the c ringproton of a conservedaromatic residue, which could interact with the agonist rr-electrondensity at its electronegative centre; (c) the amino acid at position 6, involvedin selectiverecognition, and (d) a hydrogen bond between the electronegativeatom of the rr-electron systemof the agonist andacomplementary group in the receptor, which may be important in the high affinitybinding of agonists. The authors suggestthat the invariant aspartate residueat position 11could be involvedin the establishmentof a conservedhydrogen bond. Results from this work allow us to conclude that His 134,located at position 7 of the cys-loop, plays an important role in the binding of c+BgTxto the nAChR and that it could be involvedin critical ionic or hydrogen-bondinginteractions. Acknowledgements—This work was supported in part by

grants from the Universidadde BuenosAires and the ConsejoNacional de InvestigacionesCientificasy T&cnicasde la RepirblicaArgentina (CONICET).We are indebtedto Miss Dora M. Beatti for excellenttechnicalassistance. REFERENCES

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Donnelly-Roberts, D. L. and Lentz, T. L. (1991)Binding sites for cc-Bungarotoxinand noncompetitive inhibitor phencyclidineon a synthetic peptide comprisingresidues 172–227of the a-subunit of the nicotinic acetylcholine receptor. Biochemistry 30, 74847491. Fulachier, M. H., Mourier, G., Cotton, J., Servent, D. and M&nez,A. (1994)Interaction of protein ligandswithreceptorfragments, On the residuesofcuraremimetictoxinsthat recognize fragments 128–142and 185–199of the alpha subunitof the nicotinicacetylcholinereceptor. FEBS Lett. 338,331-338.

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