Key histidine residues in the nicotinic acetylcholine receptor

~ Pergamon

0197-0186(95)00055-4)

Neurochem. Int. Vol. 28, No. l, pp. 77 87, 1996 Copyright ¢~- 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0197~0186/96 $9.50 + 0.00

KEY HISTIDINE R E S I D U E S IN THE NICOTINIC A C E T Y L C H O L I N E RECEPTOR* H. D A N I E L L A C O R A Z Z A t , M A R C E L A S. O T E R O de B E N G T S S O N a n d M I R T H A J. B I S C O G L I O de J I M I ~ N E Z B O N I N O ~ Instituto de Quimica y Fisicoquimica Biol6gicas, (UBA-CONICET), Facultad de Farmacia y Bioquimica, Junin 956 (1113), Buenos Aires, Argentina ( Received 10 June 1994 ; accepted 21 March 1995) Abstrae~Reactivity of histidine residues of the Discopyge tschudii nicotinic acetylcholine receptor was studied by reaction with DEP and the influence of their modification on functional properties of the receptor was evaluated. Determination of two kinetically distinguishable classes was achieved. The fastreacting class is composed of 7 histidine residues with an apparent velocity constant k~ = 0.0248 +0.0031 min '. The second includes at least---21 histidine residues with a velocity constant k2 = 0.0016+0.0009 min ~. The circular dichroism spectra of the native receptor and the most DEP-derivative indicate no significant modifications in the a-helix content, and fourth derivative spectroscopy analyses show that the environment around the aromatic amino acids remains unchanged. DEP treatment of the receptor results in a time- and reagent concentration-dependent loss of its ~-bungarotoxin binding ability: these results agree with those obtained with the membrane-bound receptor. The decrease in the neurotoxin binding capacity was correlated with the DEP-reaction extent of the slow groups. Incorporation of 1.93 _+0.23~nol of DEP accounted for the maximal binding capacity drop, thus indicating the involvement of two histidine residues per ~-bungarotoxin binding site. Neither amino groups nor tyrosine residues were modified during the reaction with DEP, indicating that the derivatization of histidine residues is responsible for the observed effect. Faster-reacting residues appear to be involved in agonist-induced ion flux through the nAChR channel. These results strongly support the connection between histidine residues and the receptor functional activity and lead us to infer that the changes observed in ~-bungarotoxin binding and ionic channel capacity are the consequence of independent events induced by reaction with DEP.

The n A C h R is a ligand-gated channel t h a t mediates signalling in the vertebrate n e u r o m u s c u l a r j u n c t i o n and the electroplaques of electric fish. Structural a n d

functional properties o f the T o r p e d o - n A C h R have been extensively studied since it can be o b t a i n e d in large a m o u n t s . The m a m m a l i a n muscle n A C h R shares biochemical a n d biophysical properties similar to those of the Torpedo-AChR. O u r model is the n A C h R from Discopyge tschudii--a local marine r a y f i s h - - w h i c h we purified, characterized a n d reconstituted into liposomes. Chemical characteristics a n d functional activity showed t h a t this receptor is similar to t h a t from Torpedo californica (Ochoa et al., 1983). The muscle-type receptors involve five highly h o m ologous subunits, a r r a n g e d a r o u n d a central ion pore (Zingsheim et al., 1982; Unwin, 1989; Changeux, 1990) with a stoichiometry of ct2fl76. R e c o m b i n a n t D N A technology has led to the identification of a m i n o acid sequences from several m e m b e r s of the superfamily o f ligand-gated ion channels, such as the n A C h R (see review by N o d a , 1989). A t the present time, the structure has been resolved at 9 A resolution (Unwin, 1993). Cysteines cd92 a n d ~193 were s h o w n as the M B T A

* This work was supported in part by grants from the Universidad de Buenos Aires. M.J.B. de J.B. is Career Investigator from the Consejo Nacional de Investigaciones Cientificas y TOcnicas de" la Repftblica Arqentina (CONICET). H.D.L. and M.S.O. de B. are fellows of the Universidad de Buenos Aires and CONICET, respectively. At the present time, H.D.L. is a Visiting Fellow at the National Institutes of Health. t Present address: National Institutes of Health, National Eye Institute, Genetics and Molecular Immunology Section, Laboratory of Immunology, BIdg 10 Room 10N116, Bethesda, MD 20892, U.S.A. + Author to whom all correspondence should be addressed. Abbreviations: c~-BgTx, e-bungarotoxin: Carb, carbamylcholine; CD, circular dichroism; DDF, p(N,N)dimethyl-aminobenzene diazonium fluoroborate; DEP, diethyl pyrocarbonate or ethoxyformic anhydride: MBTA, 4-(N-maleimido)-benzyltrimethylammonium ; nAChR, nicotinic acetylcholine receptor, TNBS, 2,4,6trinitrobenzene sulfonic acid. 77

78

H. Daniel Lacorazza et al.

affinity-label sites when the receptor was previously reduced (Kao et al., 1984). These cysteines are linked by a disulfide bridge (Kao and Karlin, 1986; Mosckovitz and Gershoni, 1988). Other residues have also been identified by photoaffinity labeling: Tyr 93, Trp 149, Tyr 190, Tyr 198, all of them belonging to the ~-subunit (Dennis et al., 1986, 1988 ; Galzi et al., 1990; Middleton and Cohen, 1991). In addition, a-neurotoxins, bind to the muscle-type n A C h R with subnanomolar affinity and competitively block the depolarizing action of acetylcholine ; which is why they are extensively used as binding-site labels (see review by Stroud et al., 1990). Studies based on the binding of snake a-toxins to a-subunit fragments (McCormick and Atassi, 1984; Wilson et al., 1985; Criado et al., 1986; Oblas et al., 1986; N e w m a n n et al., 1986; Mulac-Jericevic and Atassi, 1987a, b; Griesman et al., 1990; Ruan et al., 1991), synthetic peptides (Mulac-Jericevic and Atassi, 1986; Ralston et al., 1987 ; Radding et al., 1988 : Wilson and Lentz, 1988; Fulachier et al., 1994), deletion mutants (Barkas et al., 1987), or a-subunit fragments expressed in Escherichia coli transformants (Gershoni, 1987), and site-directed mutagenesis experiments (Mishina et al., 1985 ; Galzi et al., 1991 ; Tomaselli et al., 1991 ; O'Leary and White, 1992) show that the region containing Cys 192 and Cys 193 participates in the atoxin binding site of the a-subunit, apart from interacting with cholinergic ligands. It has been proposed that multiple domains of the receptor primary structure participate in the functional organization of the a-toxin binding area (Walkinshow et al., 1981). Comparison of the a-subunit sequences of the several members of the muscle-type n A C h R family allows identification of a number of highly conserved histidine residues in the ~-subunit extracellular domain. Both histidine ~-134 in the cystein-loop 128142 and histidine ~-186 close to the Cys 192 and 193 disulfide bridge are conserved in the muscle nAChRs. Moreover, when comparing the primary structure of /~, y and 6 subunits from the Torpedo californica n A C h R , it is evident that most of the conserved histidine residues in the a-subunit of muscle-type receptors are not present in the other subunits. In addition, only the a-subunit is able to bind ~-neurotoxins when receptor subunits are isolated (Haggerty and Froehner, 1981). In a previous study, we described (Lacorazza et al., 1992) the effect of DEP-treatment of the membranebound acetylcholine receptor from Discopyge tschudii on its ~-BgTx binding, and concluded that histidine residues have a major role in this ligand-recognition site. In this work, we solubilized, purified and chemi-

cally modified the n A C h R in order to determine the number of the histidine residues involved in the receptor-toxin interaction and their reactivity. In addition, we explored the influence of histidine derivatization with D E P on receptor ionic channel properties.

EXPERIMENTAL PROCEDURES

Materials The Discopyge tschudii electric organs were kindly provided by Dr Barrantes (INIBIBB, Bahia Blanca, Argentina). ~-Bungarotoxin from Bungarus multicinctus, DEP, L-c~-phosphatidylcholine (L-a-Lecithin) from soy beans, 2-4-6-trinitrobenzenesulfonic acid, Dowex WX8 (mesh 50) and Carb were obtained from Sigma Chemical Co., U.S.A. ; Affi-Gel 401 was from Bio-Rad, Richmond, Calif., U.S.A., the carrier-free ~25INa from the 'Comisi6n Nacional de Energia At6mica', Buenos Aires, Argentina and the 86Rb from New England Nuclear Corporation, Boston, Mass., U.S.A. All other reagents used were A.R. grade. Extraction and purification o f the receptor Purification was performed as described by Ochoa et al. (1983). In brief, membranes were diluted (protein concentration : 2 mg/ml) with buffer 10 mM Tris-HC1, 100 mM NaCI, 0.1 mM EDTA, 0.02% NAN3, pH 7.4 (Buffer A) supplemented with sodium cholate to achieve a final detergent concentration of 1%. After centrifugation, the supernatant was applied directly onto a 25 ml affinity column prepared from Affi-Gel 401 and bromoacetylcholine (Reynolds and Karlin, 1978). The column was previously equilibrated with Buffer A containing 1% sodium cholate and 2 mg/ml soybean lecithin. The column was washed with 5 column volumes of Buffer A containing 1% sodium cholate and 2 mg/ml lipid; the nAChR was eluted with 50 ml of 10 mM Carb in the same buffer and the eluate was immediately dialyzed against Buffer A and stored at -70°C. DEP-treatment The reagent DEP introduces a carbethoxy group (ethoxyformyl group) in the imidazole ring of histidine residues, In the present work, we will refer to the reaction with DEP as DEP-treatment. For kinetic and spectroscopic studies, the nAChR (0.1 mg/ml in Buffer A) was solubilized by the addition of 1% sodium cholate. The reaction with DEP was carried out in the cell of the spectrophotometer. Two microliters of a 35 mM solution of DEP in ethanol were added to 2 ml of the protein solution. The absorbance increase at 242 nm was recorded; readings were taken every 15 s until a plateau was reached. Measurements were performed at 25°C. The DEP-reaction extent was calculated assuming that the molar extinction coefficient of carbethoxy-imidazol in proteins is 3.9 × 103 M ~cm ~at 242 nm (Choong et al., 1977). Data analysis was carried out by utilizing a non-linear curve fitting program. For binding assays, a nAChR solution (0.2 mg/ml in Buffer A) was modified by the DEP final concentration indicated for each particular case and the reaction was stopped at predefined times by the addition of histidine until a 50 mM final concentration was reached.

Key histidines in the nicotinic receptor

Trinitrophenylation of amino groups The native and DEP-treated nAChR were treated with trinitrobenzenesulfonic acid as described by Habeeb (1966) ; proteins were dissolved (0.16 mg/ml) in 0.1 M sodium phosphate buffer, pH 7.4, containing 1% sodium cholate and TNBS, dissolved in the same buffer (final concentration: 51 mM) was added. The absorbance increase at 345 nm was monitored until a plateau was reached and the extent of the reaction was estimated by using a molar extinction coefficient of 11,500 M -I cm -1. When the effect of trinitrophenylation on the c~-BgTxbinding capacity was evaluated, a TNBS solution in 0.1 M sodium phosphate buffer, pH 7.4 (final concentration: 0.7, 1.4, 2.2 or 5.6 mM), was added to the nAChR dissolved in the same buffer (0.4 mg/ml). After 30 rain of trinitrophenylation, the reaction was stopped by adding lysine until a 57 mM final concentration was achieved, after which the binding capacity was determined.

~-Bungarotoxin iodination c~-BgTx iodination was performed by the Chloramine-T method according to Lacorazza et al. (1992). The specific activity of the monoiodinated cc-BgTx was 10 #Ci//tg.

Equilibrium binding of leSl ct-bungarotoxin 725I ~-BgTx binding was measured basically according to the procedure described by Schmidt and Raftery (1973) ;25 pmol of sites were incubated with 10-15 pmol ~25I~-BgTx in 200 /~1 of 10 mM MOPS buffer, 100 mM NaCI, 0.2% Triton X-100, pH 7.4 (NMT100) for 60 min; the mixture was then filtered onto two DEAE-Cellulose disks (DE81 Whatman) and they, in turn, were washed twice with 5 ml of 10 mM MOPS, 10 mM NaCI, 0.2% Triton X-100, pH 7.4. Typically, toxin binding reached around 1.6-1.8 pmol J251~BgTx/pmol nAChR.

Kinetics of ~-BgTx binding inhibition DEP-treatment of the nAChR (0.2 mg/ml) with different DEP final concentrations (1.8, 2.4, 3.0, 3.6 and 4.8 mM) was performed as described above. The inhibition process may be assumed as : TBS=nI

79

cation exchange column method (Gasko et al., 1976) and utilizing 86Rb as the cation (6.9 Ci per g). The assay was carried out as indicated by Medrano et al. (1987). The response was obtained from the difference between cpm Carb ÷ (in the presence of Carb) and cpm Carb (in the absence of the ligand).

Circular dichroism measurements CD measurements were performed at 25°C in a Jasco J20 spectropolarimeter. Protein concentration was 0.05 mg/ml in Buffer A containing 1% sodium cholate.

Fourth-derivate spectrophotometry analyses The fourth-derivate spectrophotometry analyses were carried out in a UV-265 Shimadzu spectrophotometer, between 230 and 350 nm at 25°C. Protein concentration was 0.05 mg/ml in Buffer A containing 1% sodium cholate. Fourth order spectra were obtained by having the built-in derivate accessory perform two successive second derivate operations with a slit width of 2 nm and 1 nm for the first and second step, respectively.

SDS gel electrophoresis SDS-PAGE was performed according to Laemmli (1970). The separating gel contained 0.1% SDS, 12.5 % acrylamide, in 25 mM Tris-192 mM Glycine, pH 8.3. The stacking gel contained 0.1% SDS, 5% acrylamide in 80 mM Tris-HC1 buffer, pH 6.8. Samples and molecular weight markers were dissolved with buffer 80 mM Tris-HCl, pH 6.8/2% SDS/10% glycerol/0.02% bromophenol blue/5% fl-mercaptoethanol. The gel was run at 15 mA.

Protein concentration This was measured according to Lowry et al. (1951). For kinetic studies, protein concentration was determined by amino acid analysis.

Amino acid analyses Samples were hydrolyzed in vacuum-sealed tubes at 110°C for 20 h in constant boiling HCI containing phenol (1 mg/ml). The hydrolysates were analyzed in a Beckman 119 CL amino acid analyser.

k, , T B S - I n

where TBS, I, TBS-In, n and k~ represent a toxin binding site, the inhibitor (DEP), the inhibited toxin binding site, the number of molecules of inhibitor reacting per a-BgTx binding site and the second order rate constant, respectively. Since, under our experimental conditions, the reaction between the nAChR and the reagent is essentially irreversible, the reverse reaction need not be considered. Although the reaction is bimolecular, it exhibits pseudo first order kinetics with the corresponding first-order rate constant k' ( = k~[TBS]). Then, the following equation may be used : log t, = log k ' + n log [DEP]. As ~BgTx-binding inhibition velocity we used the reciprocal of the half time of c~-BgTx binding inhibition (to s), which is proportional to 'v' and can be easily determined from the pseudo-first order plot.

S6Rh influx assay of reconstituted receptor Reconstitution of receptor activity was performed by the method of Dalziel et al. (1980) as modified by Ochoa et al. (1983). The Carb-catalyzed cation influx was assayed by using a

RESULTS

Kinetic study

of the reaction

As purity criteria, the purified receptor was submitted to SDS-polyacrylamide gel electrophoresis (Fig. 1). The c o r r e s p o n d i n g p a t t e r n agrees with t h a t previously s h o w n for the Discopyye tschudii nicotinic receptor (Ochoa et al., 1983). F o u r b a n d s with a p p a r ent molecular weights of 39,000, 51,000, 59,000 a n d 64,000 are present. Moreover, their a m i n o acid composition fits well with those from Torpedo californica (Table 1). The h o m o l o g y between Torpedo californica a n d Discopyye tsehudii allows us to correlate o u r results with the well k n o w n n A C h R protein-structure from Torpedo. Progress o f the receptor reaction with D E P (protein c o n c e n t r a t i o n : 70 #g/ml), at p H 7.4 a n d 25°C, is

80

H. Daniel Lacorazza et al.

-- 66.0

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(kDa) Fig. 1. SDS-PAGE ofnAChR from Discopyge tschudii, c¢, ];, 7 and 6 subunits are indicated. Molecular mass of standards appear on the right in kDa. shown in Fig. 2, a 35 ,uM D E P final concentration was used. A b s o r b a n c e increase at 242 nm was measured every 15 s until a plateau was reached. The m a x i m u m a b s o r b a n c e at 242 nm, attained after approximately 20 min of reaction, corresponds to the carbethoxylation of 28 reactive imidazole groups per m o n o m e r o f the receptor molecule (Mr. 260,000). Analysis of the data was performed by using a nonlinear curve fitting program. A good fit was obtained with a m i n i m u m of two simultaneous pseudo-first order equations with a p p a r e n t velocity constants /,h: 0.0248_+0.0031 rain ~ (Curve A) and /,~: 0.0016-+0.0009 min ~ (Curve B), that correspond to 6 . 9 + 0 . 3 , and 21.6-+0.2 carbethoxy-histidine per molecule, respectively. W h e n the modification reaction was performed under exactly the same experimental conditions but using a double D E P / p r o t e i n ratio, the study of progress of the reaction with D E P indicated Table I. Amino acid composition of nAChR from DLscop.l~qe t.schudii Residue Asp Thr Ser Glu Pro Gly Ala Val Met

Mol% 11.8+ 1.0 (11.4) 7.0_+ 1.0 (6.2) 8.0+ 1.0 (6.3) 10.0± 1.8 (10.3) 4.7±0.6 (5.7) 6.4-+ 0,9 (4.9) 5,2_+0.7 (5.2) 5.8+ 1.3 (7.5) N.D. (I.9)

Residue lie Leu Tyr Phc His Lys Arg Cys* Trp

Mol% 6,8±0.9 (7.6) 92_+0.8 (9.3) 3.4±0.5 (3.8) 4.2_+0.3 (5.0) 1.8+0.5 (2.6) N.D. (5.7) 4.0_+0.4 (4.1) 0.9+0.5 (I.3) N D . (I.5)

Values correspond to the m e a n i s t a n d a r d deviation (SD) (average of 9 determinations). N.D. : not determined. * Determined as cysteic acid. Values in brackets correspond to the amino acid composition of nAChR from Torpedt~ cali[~rnic~t (Vandlcn et al., 1979).

10 TIME

15--

(rain)

Fig. 2. Kinetic study of the reaction. Time course of the reaction of DEP (35/~M final concentration) with the histidine residues in nAChR (70/~g/ml in Buffer A containing I% sodium cholate) at 25C. Maximum absorbance at 242 nm corresponds to the derivatization of 28 imidazole groups per molecule of the nAChR. A and B are the curves calculated from experimental data by using a non-linear regression program, corresponding to two hypothetical pseudo first-order reactions occurring simultaneously. Curves A and B were calculated for k = 0.0248_+0.0031 rain J and k = 0.0016_+0.0009 min J. that, while the n u m b e r of faster residues remained invariable ( 7 . 3 ± 0 . 7 ) , the slower ones increased (32.8_+0.6). Both the UV spectrum of the DEP-treated receptor and that of the native protein were recorded. As expected, a new a b s o r b a n c e m a x i m u m was detected in the difference spectrum at 242 nm as a consequence of the introduction of a carbethoxy group in the imidazole ring of histidine residues (Fig. 3A). Furthermore, the u n c h a n g e d a b s o r b a n c e of the protein at 278 nm showed t h a t tyrosine residues h a d not been modified since their derivatization with D E P would lead to an O-carbethoxy derivative showing a decrease in a b s o r b a n c e at such wavelength (Miles, 1977). Co~!/ormational analt'sis C D spectrum of the D E P - t r e a t e d n A C h R (20 carbethoxy-imidazol groups per molecule), a n d that corresponding to the native protein were superimposable (Fig. 4) ; the a m o u n t of material prevented C D analysis of the a r o m a t i c group environment. Therefore, the fourth derivate spectra were studied (Fig. 3B). Differences between 240 and 245 n m due to DEPtreatment of the histidine residues were detected ; neither the wavelength for the m a x i m u m of each peak

Key histidines in the nicotinic receptor

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Fig. 3. Spectroscopic study of DEP-treated nAChR.(A) Spectra of native ( - - ) and DEP-treated nAChR (.-.) (70/~g/ml in Buffer A containing 1% sodium cholate) and difference spectrum (---); the reference cuvette contained the protein with the same volume of ethanol used in the DEP-solution whereas the experimental cuvette contained the DEP-modified protein solution. (B) Fourth derivate spectra of native ( - - ) and DEP-treated (---) nAChR (50 ~g/ml in Buffer A containing 1% sodium cholate (see Experimental Procedures). The modified protein contained 20 earbethoxy-imidazol groups per molecule. All the spectra were recorded at 25°C, and represent an average of 5 spectra. ÷5

order to study the involvement of histidine residues in this neurotoxin binding site (Fig. 5); absorbance increase at 242 nm represents the modification extent, whereas binding inhibition is expressed as a percentage of native binding capacity. As shown in Fig. 5, treatment with D E P results in a binding ability decrease, 52% being the m a x i m u m inhibition achieved when D E P final concentration is I. 15 m M and the DEP-reaction reaching a plateau after 12 min. This kinetic study is not comparable with the one shown in Fig. I since the conditions were not exactly the same.

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Fig. 4. Circular dichroism spectra of native ( - - ) and modified nAChR (---). The samples were dissolved in Buffer A containing 1% sodium cholate. The modified protein (50 ~g/ml) contained 20 carbethoxy-imidazol groups per molecule. The spectra were recorded at 25°C, in duplicate. nor the R parameter value was altered by histidine modification.

Effect of DEP-treatment on the ~-bungarotoxin binding The reaction of D E P with n A C h R was performed and specific '25I ~-BgTx binding was determined in

Reaction selectivity In addition to histidine residues, lysine and tyrosine could also be modified under experimental conditions. Therefore, we had to evaluate whether the ~-BgTx binding decrease was the consequence of lysine and/or tyrosine modification. The unchanged absorbance of the protein at 278 nm (Fig. 3A) during DEP-treatment, in addition to the data obtained from the fourth derivate spectra, showed that tyrosine residues had not been modified. In addition, reaction of the receptor with TNBS, a specific reagent for e and a-amino groups, was performed. Both the native and the D E P treated protein revealed no differences as regards

H. Daniel Lacorazza et al.

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Fig. 5. Histidine modification and x-BgTx binding inhibition. The receptor (80 lig/ml in Buffer A) was treated with DEP 1.15 raM. Progress of the reaction was followed by measuring the absorbance increase at 242 nm. A and B were calculated as indicated in Fig. 2. The reaction was stopped at pre-defined times by addition of a histidine excess (80 mM final concentration). The t251 :¢-BgTx binding capacity was determined as indicated in Experimental Procedures.

their incorporation of trinitrophenyl groups. Furthermore, the effect of trinitrophenylation on the L'51 ~-BgTx binding capacity of the n A C h R was assessed. TNBS-treatment (6 m M final concentration) of the receptor (0.2 mg/ml) did not significantly alter L~51~BgTx binding (data not shown).

Tirne-dependence ()/ 1:51 ~-ByTx binding inhibition hy reaction with D E P As described above, receptor ~-BgTx binding was inhibited by DEP-treatment in a time-dependent manner. In addition, the inhibition process was dependent on D E P concentration and followed pseudo-first order kinetics up to 40% of the residual L~sI ~-BgTx binding capacity (Fig. 6). This behavior could be the consequence of the modification of one or more histidine residues belonging to the same reacting family. The slope value from a logarithmic plot of the pseudo-first order rate (or t05) vs DEP concentration (Fig. 7), indicated that a minimum of 1.93+0.23 (n = 3) DEP molecules per ~-BgTx binding site accounted for the ~:5I ~-BgTx binding decrease.

EffkJcts q/DEP-treatment on channel capaciO, Both the native and the DEP-treated n A C h R s were reconstituted into soybean lecithin liposomes. Application of the method of Dalziel et al. (1980) as modified by Ochoa et al. (1983) typically yields about 75% of properly-oriented receptors. The percentage was estimated by determining the J-~5I~-BgTx binding in

the presence and absence of Triton X-100. In this configuration (right side out) the extracytoplasmic domain of receptors inserted in the liposomes is exposed to the solvent. In addition, freeze-thawing of lOO

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5 10 TIME ( m l n ) Fig. 6. Time-dependence of '251 ~-BgTx binding inhibition by DEP-treatment. The nAChR (200 #g/ml in Buffer A) was treated with 1.8 mM (O), 2.4 mM (A), 3.0 mM (C)), 3.6 mM ( ~ ) and 4.8 mM ( i ) DEP final concentration. At predefined times the reaction was stopped by addition of a histidine excess (50 mM final concentration). Data were fitted to a straight line by using a linear regression program.

Key histidines in the nicotinic receptor

83 DISCUSSION

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Fig. 7. N u m b e r of D E P molecules interacting with the n A C h R in the ~-BgTx i n h i b i t i o n process. Values in the p l o t were t a k e n f r o m Fig. 6. D a t a were fitted to a straight line by using a l i n e a r regression p r o g r a m . Slope : 1.93 _ 0.23.

the reconstituted vesicles increases both the agonistinduced fluxes and the vesicle internal volume, as previously reported (Ochoa et al., 1983). Table 2 shows that the reconstituted nAchR exhibits a Carb-catalized cation influx, thus indicating that it is functional. The table also shows results of the 86Rb + influx assay with the DEP-treated nAChR. The ligand-gated ion channel capacity was drastically inhibited when only 6 ethoxyformyl groups per nAChR molecule were introduced--the response being 25% of that of the unmodified protein ; nevertheless, this derivative accounted for 80% of the native toxin binding ability. When 10 carbethoxy groups were incorporated, the cation influx was completely lost while the toxin binding ability dropped to 62%. Even after modification of 28 histidine residues, the protein still had a residual binding capacity.

The Diseopyge tschudii nicotinic receptor was submitted to SDS-polyacrylamide gel electrophoresis and four bands were present ; their molecular weight (Fig. 1) and amino acid composition (Table 1) fit well with those from Torpedo californica and the ones reported in our previous work (Ochoa et al., 1983). When the reaction of DEP with the receptor was carried out under the conditions indicated in Fig. 2, seven histidine residues were within the faster family and the remaining 21 in the slower family. The fact that the same number of histidine residues was found in the faster family when a higher DEP/protein ratio was used indicates that probably only 7 residues compose the faster group. The u.v. spectrum of the DEPtreated receptor and that of the native protein showed no changes except for the introduction of the carbethoxy group in the histidine imidazolic ring thus indicating the absence of modifications different from those of histidine residues. As judged by the far-ultraviolet CD spectrum of the DEP-treated nAChR (20 carbethoxy-imidazole groups per molecule), when compared with the corresponding spectrum of the native protein (Fig. 4), the a-helix content had not been altered by reaction with DEP. However, fourth-derivative spectroscopy --which can be carried out with a minimum protein amount--was used to further investigate possible conformational modifications in the modified protein. Changes in the environment of the aromatic residues led to changes in the fourth-derivate spectra thus reflecting the alterations suffered by the electronic energy levels (Donovan, 1969 ; Laskowski, 1970). This was specially important in this case since DEP can modify lysine and tyrosine, in addition to histidine residues. In order to quantify the changes, we used the wavelengths of the maxima and minima, and a geometrical parameter R defined as the ratio between the amplitude of the two main peaks (Padr6s et al., 1982; Dufiach et al., 1983). The spectra (Fig. 3B) indicated that neither the wavelength for the

Table 2. Effect of nAChR DEP-treatment on its ligand-gated ion channel capacity 86Rb+ Influx Experimentalconditions Native nACh R DEP-nAChR 6* 10 14 28

Carb + (cpm) 2432 + 45 1029_+30 518_+25 1044-+47 1426_+76

Carb

(cpm)

686 + 69 589_+6 521 +33 1003_+ 135 1286_+ 168

Response (%)

'251 ~-BgTx binding nmot/mg protein (%)

100 25 0 0 0

100t 80+ 14 62_+7 39_+5 27_+ I

* Number of histidine residues modified per nAChR molecule. Values represent mean _+S.D. of experiments in triplicate. t 5.8 + 0.6 nmol '25I ct-BgTx/mg of protein.

84

H. Daniel Lacorazza et al.

maximum of each peak nor the R parameter value (data not shown) was modified thus indicating lack of conformational modifications in the tyrosine, tryptophan and phenylalanine environments. This finding, added to the absence of changes in absorbance at 278 nm in the u.v. spectrum, indicated that tyrosinc residues had not been modified. On the other hand, reaction of nAChR with TNBS, a specific reagent for and ~: amino groups, revealed that amino groups had not been modified either: additionally, TNBStreatment of the receptor did not alter its ~-BgTx binding capacity. These data, as a whole, allow us to conclude that only histidine residues are involved in the DEP binding inhibition process. Our results lead us to infer the existence of two key histidine residues per ~-BgTx binding site. However, the increase in the DEP-reaction extent, over 28 carbethoxy-histidine residues per molecule, does not lead to a further :~-BgTx binding drop ; therefore, the possibility of a binding site population not being carbethoxylated or of one whose derivatization does not bring about a toxin binding decrease, cannot be discarded. This evidence agrees with previous results obtained with the membrane-bound receptor (Lacorazza et al., 1992) and benzodiazepine receptor (Maksay and Ticku, 1984). As regards DEP-treatment effects on the x-BgTx, results obtained in this work agree with those reported after DEP-treatment of the membrane-bound receptor (Lacorazza et al., 1992). In that system, DEPtreatment also inhibited the toxin binding ability and, consistent with the modification of a binding site, it did not alter the affinity but reduced the number of receptors. Moreover, comparison of the evolution of the inhibition curve with that of curve B (Fig. 5) leads us to suggest that chemical modification of histidine residues belonging to the slower family might be responsible for the toxin binding decrease the toxin binding ability remains practically unchanged even after all residues in the faster family have been modified. Other key histidine residues belonging to slow reacting farnilies have been reported, either in enzymatic proteins such as horseradish peroxidase (Bhattacharyya et al., 1992) and chloroperoxidase (Blanke and Hager, 1990) or in hormonal proteins as human growth hormone (Fukushima et al., 1990). The above results suggest that there may be some kind of noncovalent interaction between the key histidine residues and other sites in the molecule, or that such residues are only partially exposed to the solvent. On the basis of :~-BgTx structure analysis, Love and Stroud (1986) have proposed that the interface between the nAChR and the toxin primarily involves hydrophobic and

hydrogen-binding interactions in addition to electrostatic ones such as those of Lys 184 and Arg 187 of the u,~ subunit which are critical for ~-BgTx binding (McLane el al., 1991). Even if no histidine residues had a direct participation in the binding but one or more of them were in the molecular region involved, their modification might hinder or even prevent such binding. McCarthy and Stroud (1989) have shown -by using tritiumhydrogen exchange---that interaction between the Torpedo receptor and ~-BgTx evokes a change in secondary structure thus suggesting that receptor chains re-form a new/4-sheet pattern with those of the toxin molecule. The toxin binding decrease induced by DEP-treatment leads us to infer that the steric perturbation of the carbethoxy group introduced in the imidazole ring may alter such conformational arrangement. Moreover, it has been shown that the recepto> toxin interaction is associated with changes in the fluorescence spectrum (Kang and Maelicke, 1980; Johnson el al., 1984; Cheung el al., 1984) and, in a less complex system, an increase in fi-structure upon ~-BgTx binding to the Torpedo cal(lbrnica ~ 181 200 peptide becomes evident (Conti-Tronconi e t a / . , 1991). Finally, comparison of the data from Table 2 allows us to conclude that there are key histidine residues involved in both processes : ion channel capacity and toxin binding ability, the former being more responsive to DEP-treatment. It must be pointed out that cation influx is gated by Carb; therefore alterations in the ~-BgTx binding site need not affect the colinergic site. This result agrees with those obtained by histidine modification of BC3H1 cells with DEP followed by analysis of channel property by patch clamp (Bouzat el al., 1993). In addition, results strongly suggest that histidine residues involved in the receptor ion fluxing ability, belong to the fast-reacting family, thus indicating they are not the same as those which participate in the toxin binding site. Furthermore, results support our proposal that the effects described are the consequence of independent events induced by DEP-treatment. With regard to the possible topography of the histidine residues involved, given the known primary structure of Torpedo receptor, and taking the consensus l~ur-transmembrane model (Noda et al., 1989 and references therein) the following remarks become evident: (i) the extracellular domain includes the large N-terminal moiety containing 8, 4, 4, and 7 histidine residues in the :< fi, 7, and 6 subunits, respectively: the loop between hydrophobic segments M2 and M . with 2 histidine residues in the ~-subunit, and the C-

Key histidines in the nicotinic receptor terminal, with n o histidine residues. This m a k e s a total o f 33 histidine residues in the extra-cytoplasmic d o m a i n of Torpedo californica nicotinic acetylcholine receptor. As the experiments were performed with detergent solubilized receptor 2, 1, 1, a n d 3 histidine residues could be exposed to the cytoplasmic d o m a i n in the ~, fl, y, a n d c5 subunits, respectively. These 7 histidine residues are potentially modifiable by D E P but its derivatization is n o t likely to produce a n altered ctBgTx binding in the N - t e r m i n a l of the molecule. C o m p a r i n g the a m i n o acid sequences o f the k n o w n subunits by h o m o l o g y , it is clear t h a t histidine residues ~- 134 a n d ct- 186 are conserved in the muscle receptors. The histidine ct-186 has been s h o w n to be critical for ~BgTx b i n d i n g in mutagenesis directed experiments ( C h a t u r v e d i et al., 1993). We are currently p e r f o r m i n g experiments in order to localize the key histidine residues whose derivatization with D E P leads to a n altered ctBgTx binding capacity. Acknowledgements--We thank Dr Alejandro C. Paladini for performing CD spectra and for critical reading of the manuscript. We are indebted to Miss Dora M. Beatti for excellent technical assistance and Professor Rex Davis for language supervision of the manuscript. REFERENCES

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