Neurochem. Int. Vol. 20, No. 4, pp. 521 527,1992 Printed in Great Britain.All rights reserved

0197-0186/9255.00+0.00 Copyright~ 1992PergamonPress Ltd


(Received 2 May 1991; accepted 27 September 1991) Abstract--This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopy.qe tschudii on its specific ~-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrocarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 rain period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70% ; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the ~-bungarotoxin binding site of the nicotinic acetylcholine receptor.

Similarity between the receptors from the electroplaques of some electric fish (Torpedinae family) and those from mammalian muscle, and the former's ready isolation in significant amounts, makes it an ideal model for acetylcholine receptor biochemical and functional study. Researchers working in the southern cone of the western hemisphere have no easy access to either Torpedo californiea or Torpedo marmorata, two of the better studied species. We can resort, however, to Diseopyge tshudii, a Narcidinae that may be caught in the South Atlantic or Pacific waters (Norman, 1937; Ringuelet and Aramburu, 1960; Krefft and Stehman, 1973; Ochoa, 1980). In a former paper (Ochoa et al., 1983) we purified and characterized the nicotinic acetylcholine receptor from Diseopyge tshudii. Native membranes, rich in nicotinic receptors, showed carbamylcholine-catalyzed cation transport blocked by curare and desensitized by prior incubation with

cholinergic agonists. When the receptor was purified by affinity chromatography, SDS-polyacrylamide gel electrophoresis and amino acid composition of each individual subunit revealed the receptor is very similar to that from Torpedo ealiforniea. On the other hand, a new era in neurobiology began with the successful cloning and sequencing of DNA coding for subunits of the acetylcholine receptor (Claudio et al., 1983; Noda et al., 1982, 1983a, b; Sumikawa et al., 1982). These experiments are the cornerstone towards studying the relationship between the structure of these protein molecules and the electrical activity of the nervous system. In this respect, the acetylcholine binding site of the receptor was the first functional site to be covalently modified by specific chemical reagents. Karlin (1969) was able to prove the presence of a readily reducible disulfide bridge close to the negatively-charged subsite of the acetylcholine binding site. After reduction and alkylation with affinity labels, the authors concluded that the effect of a type of reagents, i.e. MBTA, results in the inhibition of the subsequent responses of the receptor to agonists while * Author to whom all correspondence should be addressed. Abbreviations: nAChR-M, nicotinic acetylcholine receptor the effect of another type of reagents, BAC, the nitromembranes; ct-BgTx, 0t-bungarotoxin; DEP, diethyl phenyl ester ofp-carboxyphenyltrimethyl ammonium pyrocarbonate ; Carb, carbamylcholine. (NPTMB) (Silman and Karlin, 1969 ; Cox et al., 1979) 521


IfUGO D. LA('ORAZZA t,,t O1.

and 3(~-bromomethyl),3'-~(trimethyl a m m o n i u m ) methyl azobencene, results in the activation o f the receptor (Bartels el al., 1971; Lester et al., 1980). Reactions o f M B T A and BAC are mutually exclusive (Damle et al., 1978) and blocked by the presence o f ligands o f the acetylcholine binding site. This reducible disulfide bridge has been identified as that involving Cys 192 and Cys 193 ( K a o el a/., 1984), two amino acids belonging only to the e-subunit (Noda el al., 1983a). Moreover, MBTA, a sulfydril-directed reagent, labels the cd79-207 cyanogen bromide fragment in native Torpedo m a r m o r a t a A C h R (Dennis el al., 1986). On the other hand, the study o f acetylcholine receptor ligand interactions has been facilitated by the use o f neurotoxins, such as :~-bungarotoxin, which bind to the receptor with high affinity. The effect o f M B T A and BAC are also blocked by these curaremimetic neurotoxins. Furthermore, Wilson et al. (1985) and N e u m a n n et al. (1986) have been able to prove that synthetic peptides corresponding to the molecular region comprising the reducible disulfide bridge c~Cys 192-Cys 193 are capable o f binding ~-toxins, although the binding does not respond to agonists and has a lower affinity than that o f native A C h R . Two histidine residues are located in the surroundings o f the relevant disulfide bridge. This report describes the effect o f ethoxyformylation o f the memb r a n e - b o u n d acetylcholine receptor from Discopy.qe tshudii on its ~-bungarotoxin binding as a contribution to the knowledge on its ligand recognition sites.


MateriaLs Electric fish : live male and female Discopyge tshudii specimens were caught near the port of Mar del Plata and sent by air-freight in sealed polythene bags containing 02 and sea water. On arrival, the fish were killed by pithing and dissected electric organs weighed and stored in liquid nitrogen. ~-bungarotoxin from Bungarus multicinctus and carbamylcholine were obtained from Sigma Chemical Co., U.S.A. and the carrier free '2SINa from the "Comisidn Nacional de Energia Atdmica". All other reagents used were A.R. grade. The acetytcholine receptor enriched membrane fraction was prepared according to Ochoa et al. (1983). Acetylcholinesterase activity was measured by the method of Ellman et al. (1961). ~-bungarotoxin iodination This was performed by using Chloramine-T method. Briefly, 10 nmol of ~-BgTx were dissolved in 50 #1 of 0. I M phosphate buffer, pH 7.4 and 0.5 mCi of carrier-free ~2qNa was added : right after that the addition of 5 nmol of Chloramine-T in 5/tl of distilled water was effected. The reaction

developed for 2 min at room temperature and was stopped by adding 6 nmol of sodium metabisulfitc in 2/d of distilled water and by dilution with 70 ill of the same phosphate buffer. The reaction mixture was chromatographed through a Sephadex G2> column (62 x 0.7 cm) equilibrated and elated with 0.1 M phosphate buffer, ptt 7.4. The fractions con-taining the 125]~-BgTx were pooled and the binding capacity was determined by using an AChR preparation purified by' alfinity chromatography.

Equilibrium binding q / " ' l It was measured according to the procedure described by Schmidt and Raftery (19731, modified as follows: 2 5 pmol of sites were incubated with 10 15 pmol ~2Sl c~-BgTx in 200 ill of buffer 10 mM MOPS, I00 mM NaC1, 0.2% Triton X100, pH 7.4 (NMTI00) for 60 min, the mixture was then tiltered 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% Tritdn X-100, pit 7.4. When equilibrium binding of ':sI ~-BgTx was measured m the presence ofcarbamylcholine, the membranes in NMT 100 were incubated at room temperature for 1 h with 20 pmol of '~51 :~-BgTx and carbamylcholine at a 10 mM final concentration. Chemical trealmenl o/ memhranes The ethoxyformylation reaction was carried out by adding DEP, freshly dissolved in ethanol, to 300 Ill of membrane suspension (0.2 mg/ml) in Tris HC1 10 mM buffer, pH 7.4, containing NaC1 100 mM, EDTA 0.1/~M and N3Na 0.02°/{, ( Buffer A). The reaction developed at room temperature for 30 rain under constant magnetic stirring. Ethanol concentration never exceeded 1% and had no effect on radioligand binding. When time-dependence of the inactivation of radioligand binding was studied, a DEP/protein ratio able to inhibit about 50% of such binding was selected (between 1 and 2 mM DEP final concentration). 150 #1 aliquots were taken from the 750 ~ll of the original reaction mixture at definite time-intervals and the reaction was stopped by adding an excess of histidine (final concentration : 35 mM) and by decreasing the temperature to 4 C. For chemical lreatment in the presence of agonist, membranes were incubated with the concentrations of ligand indicated for each particular case, in buffer A, at room temperature, for 30 rain. At the end of the incubation period, ethoxyformylation was performed as indicated above (1.6 mM DEP final concentration). Then, membranes were centrifuged at 4'C for 211 min at 18,000 rpm and the ligand removed by washing the membranes with 1 ml of the same buffer eight times. Finally, membranes were resuspended in the initial volume of buffer A and the ~51 :z-BgTx equilibrium binding was determined. Control experiments were performed in the presence of the same buffer, ligand and solvent but without DEP. When evaluating the effect of the pH medium on the ethoxyformylation reaction, membranes were twice centrifuged at 4 C for 20 rain at 18,000 rpm and resuspended in 0.1 M phosphate buffer at pH values ranging from 6 through 8. Ethoxyformylation was then carried out as described above. At the end of the modification reaction, membranes were centrifuged and resuspended m buffer A and their '-151 >BgTx binding was determined. De-etho.u~lorm)'lation q/" DEP-treated membranes Nine hundred pl nAChR-M (protein concentration : 0.24 mg/ml) were treated with DEP (1.6 mM final concentration)

Histidine residues in ct-bungarotoxin binding


for 30 min. A hydroxylamine solution was added to obtain a 0.5 M final concentration ; after definite time-intervals (Fig. 5) hydroxylamine was eliminated after a six-time membrane centrifugation (at 18,000 rpm, 4°C, 20 min) and resuspension in buffer A. SDS-yel electrophoresis It 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-HCl buffer, pH 6.8. The samples and molecular-weight markers were treated with sample buffer (2% SDS, 10% glycerol, 80 mM TrisHC1, 0.02% Bromophenol blue, 5% fl-mercaptoethanol) at 100°C for 2 min prior to electrophoresis. Protein concentration This was measured according to Lowry et aL (1951).





Data analysis Analysis of saturation and competition curves was carried out by utilizing a non-linear curve fitting program (Gauss Newton method as modified by Frazer and Suzuki, 1973).

RESULTS Figure 1 shows the SDS-polyacrylamide gel electrophoresis pattern of the three fractions obtained from Discopyye tshudii electroplax-rich membrane preparation. Gels with lower cross-linking (not shown) also revealed the presence of the higher molecular weight proteins such as ATPases, cholinesterases and a 98 kDa band, characteristic of these membranes. Fraction C - - l o c a t e d between the 35 and the 41% s u c r o s ~ w a s selected on account of its higher toxin binding value and its lower cholinesterase activity when compared with Fractions A and B (Table 1). Treatment of membranes with D E P resulted in a concentration-dependent loss of ~25I ~-BgTx binding, 70% being the maximum inhibition reached (Fig. 2). A plateau was reached at 2 m M D E P final concentration. U n d e r our experimental conditions the results could be the consequence of histidine, tyrosine or lysine modification at or near the binding site. On the other hand, it should not be discarded, beforehand, that ethoxyformylation of other histidine, tyrosine or lysine residues can be associated with molecular conformational changes responsible for the inhibition of the toxin binding. In order to ensure histidine modification, ethoxyformylation was carried out at different pH values; BgTx binding was then determined. Figure 3 shows that, as expected, ct-BgTx binding drops as pH increases from 6 to 8. Progress of the ethoxyformylation reaction at room temperature, pH 6.0 and 7.4 and D E P final concentration 1.0 m M is shown in Fig. 4. Modification is


Fig. 1. SDS-gel electrophoresis pattern of fraction C from electroplax rich membrane preparation, ct, fl, 7 and 6 are the four receptor subunits which occur in the stoichiometry ot2f175 (Reynolds and Karlin, 1978; Lindstrom et al., 1979; Raftery et al., 1980; Ochoa et al., 1983). Markers used for apparent molecular weight comparison: Albumin (bovine plasma), Ovalbumin, Pepsin (right lane).

faster at pH 7.4, the binding capacity drops dramatically at the start of the reaction and a plateau is reached after only 15 s. On the other hand, at pH 6.0, binding ability decreases only by 30% after 10 min. In order to obtain a stronger evidence of histidine participation at the bungarotoxin binding site, membrane ethoxyformylation reversion was studied. Figure 5 shows that de-ethoxyformylation of the receptor Table 1. Selectionof nAChR-membrane fractions Toxin binding (T) (pmol ~"sI- Cholinesterase(C) (~mol :tBgTx/mgof acetylthiocholine/mgof Fraction protein) protein" min) A B C

176+35 295_+31 668_+35

5.709_+0.455 3.217_+0.145 3.703_+0.311

T/C 30 92 180




I00 ,






~5 C










............................ ~

~ ........




I 2

I 3







..¢,0, ~




















Time (min) Fig. 5. Binding capacity restoration by de-ethoxyformylation of DEP-treated nAChR-M. Hydroxylamine (final concentration 0.5 M) was added to DEP-treated ( O ) and native membranes ( ( 3 ) .

correlates very well with the restoration of its c¢-BgTx binding capacity. Saturation curves of ~2~I ~-BgTx binding to the acetylcholine receptor and Scatchard analysis of the data show that B ..... value from DEP-treated membranes drops but affinity remains unchanged (Figs 6 and 7). When ethoxyformylation is carried out on 10 m M Carb previously-incubated membranes, the effect is significantly lower (Table 2). At that Carb concentration the ~-BgTx binding is almost completely inhibited.






Fig. 2. c¢-BgTx binding decrease by nAChR-M ethoxyformylation. 300 pl nAChR-M (protein concentration : 0.17 mg/ml) were treated with the indicated final concentration of DEP in ethanol, at room temperature, for 30 rain.






Fig. 3. Ethoxyformylation of nAChR-M was performed at indicated pH values. After appropriate change of pH medium, bungarotoxin binding was determined (see Experimental Procedures).


Fractionation of receptor-rich membrane preparations from Discopyge tshudii electroplax enabled us to obtain three fractions. SDS-polyacrylamide gel electrophoresis patterns of these fractions are consistent with the presence of the acetylcholine receptor and determination of the corresponding molecular weight values agrees with previous data (Ochoa et al.. 1983). Fraction C, which exhibits the higher ~-BgTx

8O "D e-




1.0 0.8








0.4 05.






Fig. 4. Ethoxyformylation of nAChR-M at pH 6.0 (-Q-) and 7.4 ( O ). After the corresponding time-intervals, reaction was stopped by adding an excess of histidine (final concentration: 35 mM). Then, :¢-BgTx binding was determined at pH 7.4.




1251o( -




BgTx (cpm




x l O -3 )

Fig. 6. Scatchard plots of ~-BgTx binding data of native (0) and 0.5 mM (O) and 1.0 mM DEP-treated ( ~ ) membranes.

Histidine residues in ~-bungarotoxin binding lOOe• 80




~ ........ 0,5




D E P (mlUl)

Fig. 7. Effect of DEP concentration o n Bmax value. The Bm,x was obtained by linear regression of the binding data. binding and the lower cholinesterase activity was used (Table 1). Membrane ethoxyformylation was carried out and specific ~25I ~-BgTx binding was determined in order to investigate the participation of histidine residues in this neurotoxin binding site. Results showed a DEP concentration-dependent loss of such recognition ability; major binding decrease is reached as a consequence of the ethoxyformylation--70% under our experimental conditions (Fig. 2). This effect may be due to histidine modification. Nevertheless, tyrosine or lysine residues could be simultaneously modified. On the other hand, the effect may be the consequence of the direct modification of the binding site or some kind of conformational change which indirectly induces binding decrease. A series of experiments was performed in order to elucidate these points. Ethoxyformylation was carried out at different pH values (Fig. 3). Among the DEP-modifiable amino acids only the histidine residue has a pK value which allows obtention of such experimental curve. If lysine or tyrosine residues were involved in the binding mechanism, at least part of the curve would be shifted to higher pH values. When ethoxyformylation was performed at pH 6.0 (Fig. 4), the modification rate was lower than that at pH 7.4 (as expected since Table 2. Effect of 10 mM Carb preincubation on membrane ethoxyformylation

Experimental conditions Native membranes DEP-treated membranes Membranes incubated with !0 mM Carb Membranes preincubated with 10 mM Carb and DEP-treated

1251 ~-BgTx binding (%) 100 37 + 2 103 + 7 55 + 5*

* P < 0.02 when compared with the binding value of DEP-treated membranes. Binding of ~25I~-BgTx was performed after washing out the ligands as indicated in Experimental Procedures.


DEP reacts with unprotonated species), thus strongly supporting such results. Furthermore, we proved there is a good correlation between binding ability restoration and de-ethoxyformylation. Selectivity of DEP is considered to be the highest for histidine at pH 6.0 (Lundblad et al., 1984) ; at pH 7.4 there is a major possibility of lysine modification but ethoxyformylation does not reverse. However, the reversal of nAChR-M histidine derivatization allows complete restoration of its binding capacity even if its modification is made at pH 7.4. These results also discard the participation of lysine residues and further support that of histidines. Scatchard analysis shows that the Bmax value from DEP-treated membranes drops but affinity remains unmodified (Figs 6 and 7). These data strongly support the finding that histidine residue/s is/are involved in the ~-bungarotoxin binding site and discard the existence of any large conformational changes. If conformational changes exist they are bound to be localized and reversible. Moreover, comparison between data from Figs 2 and 7 supports the conclusion that, those histidine residues whose ethoxyformylation leads to the binding capacity decrease, are at the toxin binding site, as the decrease in the number of receptor correlates very well with the extent of toxin binding decrease induced by ethoxyformylation. Finally, membranes were preincubated with 10 mM carbamylcholine and then ethoxyformylated, Data included in Table 2 show that after membrane DEPtreatment, 63% of the toxin binding sites is modified. When ethoxyformylation is performed after membrane incubation with 10 mM carbamylcholine, toxin binding reaches 55 % - - t h e remaining 45% of the sites being modified. This implies that 35% of the sites modifiable under our experimental conditions are protected by the agonist binding. This result is consistent with partial overlapping of the sites and, on the basis of the existence of "multiple binding sites" for the neurotoxin (Walkinshaw et al., 1981), preincubation with carbamylcholine may not protect toxin binding completely ; moreover, this is an effect to be expected on account of their very different molecular sizes. On the other hand, it is well-known that exposure to high agonist concentrations results in a desensitized state of the receptor; nevertheless, the influence of ethoxyformylation on toxin binding is evident even in that state of the receptor. From all the above, we can conclude that histidine residues are involved in the ~-BgTx binding.



Ethoxyformylation o f the benzodiazepine receptor-- which is also a m e m b e r o f the superfamily o f ligand-gated ionic c h a n n e l s - - s h o w s that a histidine residue is critical, at least, for a part o f the benzodiazepine binding sites (Butch and Ticku, 1981: Maksay and Ticku, 1984; Lambolez e t a / . , 1989). Our preliminary results on ethoxyformylation o f A C h R , purified by affinity c h r o m a t o g r a p h y , confirm the major role o f histidine residues in the toxin binding mechanism. We are now interested in defining localization o f the key residues in the primary structure of the protein. Acknowledgements The authors thank Dr E. L. M. Ochoa [or critical reading of the manuscript. This work was supported in part by grants of the Universidad de Buenos Aires and the Fundaci6n Antorchas.

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Role of histidine residues in the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific ...
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