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Proceedings of the National Academy of Sciences

Vol. 67, No. 1, pp. 74-78, September 1970

Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*
Henry G. Mautner and Eva Bartels
DEPARTMENT OF BIOCHEMISTRY AND PHARMACOLOGY, TUFTS UNIVERSITY SCHOOL OF MEDICINE, BOSTON, MASSACHUSETTS 02111, AND DEPARTMENT OF NEUROLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, NEW YORK, NEW YORK 10032

Communicated by David Nachmansohn, June 4, 1970

Abstract. p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholinereceptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or pnitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptoractivator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue.

Introduction. A considerable body of evidence has accumulated in favor of the view that acetylcholine induces a conformational change of the receptor protein in excitable membranes, thereby triggering off a series of reactions resulting in increased ion permeability.1 2 Much effort has been expended on isolating the biopolymer on which acetylcholine acts during activity.3'4 Such work would be aided by highly specific reagents suitable for labeling of the macromolecules in question by formation of covalent bonds with groups at or near the active site. In recent years, several such "active-site directed" labeling reagents have been reported.5-8 This present study is an extension of the work by Changeux et al.8 with the acetylcholine receptor and of Wofsy and Michaeli9 with acetylcholine esterase on one of these reagents, p-trimethylammonium benzene diazonium fluoroborate (TDF). The latter compound, which had been used as an affinity-labeling reagent for anti-p-azophenyl trimethylammonium antibodies, 10 is an irreversible inhibitor of the acetylcholine-receptor site of the electroplax at the level of the junctions.
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ACETYLCHOLINE RECEPTOR AND ESTERASE

75

TDF, being a diazonium salt, readily forms covalent bonds with suitable amino acid residues. The potent receptor inhibitors d-tubocurarine, dimethyl dtubocurarine, and flaxedil protect against the irreversible inhibition by TDF, even when ten times larger concentrations of TDF are used.8 In the present study we have tested TDF, its dimethyl analog (DDF), its p-nitro analog (NDF), and its p-acetoxy analog (ADF). The potencies of these compounds in affecting the monocellular electroplax preparation were compared and their stabilities in solution were determined. The relative abilities of TDFl and NDF to inhibit acetylcholinesterase were also studied. Method. All compounds were synthesized and purified by methods described ill the literature (TDF,1' DDF,12 NDF,13 ADF13).
Because the electroplax experiments were carried out in pH 6 eel Ringer's solution, studies of the decomposition of the above compounds were carried out in that solvent using 1 mg/100 ml solution of TDF and its analogs. Reaction rates were followed by observing the disappearance of ultraviolet extinction at 253 nm for TDF, 378 nm for DDF, 260 nm for NDF, and 269 nm for ADF. The compounds were placed in thermoregulated (25.00C), stoppered quartz cuvettes. A Cary model 15 recording spectrophotometer was used. Plots of log A against time yielded straight lines, from the slopes of which the first-order rate constants of decomposition were obtained (see Table 1). Single cells from the electric organ of the electric eel were dissected and mounted in a lucite chamber according to the procedure developed by Schoffeniels.14 The method of recording the resting potential was the same as that described previously."5 The composition of the eel Ringer's solution was NaCl 160, KCl 5, CaCl2 2, MgCl2 2, NaH2PO4 0.3. and Na2HPO4 1.2 mM. The incubation pH was 7.0, the temperature 250C. The compounds were dissolved in Ringer's solution at pH 6.0, with a phosphate buffer concentration of 50 mM immediately before use. In some experiments 1 mM L-histidine was added after the application of TDF and NDF, to quench unreacted molecules, but no difference in the results was observed when. the- cell was washed in Ringer's only. Results. Figure 1 shows dose:-response curves for carbamylcholine with and

without pretreatment with 0.01 mM TDF and NDF or 0.1 mM ADF for 20 min. It can be seen that TDF and ,IERinhibit the carbamylcholine response to an almost identical extent"whiMe'ADF is ten times weaker. If a cell is incubated with 0.05 mrnM TDF or NDF for 20 min, the response to 0.05 mM carbamylcholine is abolished- and 1 mM carbamylcholine causes only 5-10 mV depolarization. NDF also inhibits the depolarizations produced by tetramethylammonium and phenyltrimethylammonium. The observations of Changeux et al.8 have suggested that phenyltrimethylammonium and curare compete with TDF for the same binding sites on the receptor. Similar results have now been obtained with NDF. Hexamethonium, curare, and to a lesser degree tetramethylammonium protected the binding sites against the action of
FIG. 1.-Dose-response curves for carbamYl- E-Eo choline before and after exposure of the cell to mV p-trimethylammonium benzene diazonium fluo- 60 roborate (TDF) and its p-nitro (NDF) and p- 50 acetoxy (ADF) analogs. Two dose-response 40 *curves of carbamylcholine were obtained on a 30 o , single cell, one before and one after application of 20 either 0.01 mM TDF, 0.01 mM NDF, or 0.1 mM lol' 0 i -, ADF for 20 min. 0, El, A controls, X NDF, 5 TDF, A ADF, * 0.05 mM NDF or TDF. Eo = initial resting potential, E = steady state potencarb, 10-5 M tial in presence of carbamylcholine.

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BIOCHEMISTRY: MA UTNER AND BARTELS

Puoc. N. A. S.

NDF. As shown in Figure 2B, the reipons4 to 0.05 mM carbamyicholine was only slightly reduced if 0.05 mM NDF was applied together with 0.5 mM hexamethonium. If the cell were to be exposed to NDF alone, the response to 0.05 mM carbamylcholine would be absent (see .'Pig. 1). Hexamethonium is about half as effective in the protection of the binding sites against TDP than against NDF (Fig. 2A); this is measured by the response to carbamylcholine after exposure to 0.5 mM hexamethonium in the presence of the two analogs.
A
-mV

cccc
X

cc

'

'51
N.R.

^
N.R.

i
B

IN.ft.
72 82 87

5 60 -5xlO 0-4

60

ex

40

40 t-N.R.
0
5

t 'R
10

..
81

65

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TIME IN MINUTES

FIG. 2.-ompetition between hexamethonium and diazonium compounds. (A) After the test response to 0.05 mM carl1gmylcholine, the cell was incubated in 0.5 mM hexamethon~ium for 15 mmn folloWed by 0.5 mM hexamethonium plus 0.05 mM TDF for 20 mmn; then the cell w~s rinsed in noriial Ringer's (N.R.) for another 20 min to remove the hexametheiiium. Further applications of carbamylcholine depolarized the cell partially. (B) Same experiment as in A, except that NDF was used instead of TDF.. The,-second and third exposures to carbamylcholine give a decreased response, but riot as much as after incubation with TDF.

As reported by Podleski et al.,16 TDF becomes a very potent, reversible receptor activator after exposure of the cell to dithiothreitol, which reduces S-S bonds in the vicinity of the receptor site. 17 NDF, evren at a concentration of 1 mMI, does not depolarize the electroplax membrane after dithiothreitol, but inhibits irreversibly the depolarization caused by TDF, regardless of whether it is applied before or after dithiothreitol (Fig. 3). Mfter dithiothreitol the response to 0.04 mMd carbamylcholine is reduced, while 0.005 mM TDF causes a fast depolatization which is reversed immediately and completely on addition of 0.1 mM NDF in the presence of 0.005 mM TDF. Mfter removal of NDF from the test solution, TDF no longer depolarizes. The same effects are observed when the reduced cell is incubated in 0.05 mM NDF? for 10 mmn and washed in Ringer's: the depolarizing action of TDF is abolished. TDF is self-inhibitory. Mfter incubation of the cell in 0.1 mM TDF for 20 mmn before reduction, the depolarizing action of TDF is also abolished.

VoL. 67, 1970

ACETYLCHOLrNE RECEPTOR AND ESTERASE


-mV
90 80-

77

CC 4xO5 6

DTT IxO3
CC 4x0-5

TDF 5xIO |

TDF

5x1O 6

FIG. 3.-Antagonism between TDF and NDF after disulfide reduction by dithiothreitol. For explanation see text. CC, carbamyl choline; DTT, dithiothreitol.

70
60 -

R.
l
N.R.
TDF

50

I40

5x1O

6+NDF IxIO4
52 56

IN.R. 30L 4 8 16 28 32 36 44 48 12

TIME IN MINUTES

Discussion. The stabilities of the various analogs in eel Ringer's solution, at pH 6, decrease from DDF to TDF to NDF to ADF. The half-life of ADF is 40 min compared to 69 min for NIDF (Table 1). Since the solutions were preTABLE 1. Decomposition of p-trimethylammonium benzene fluoroborate (TDF) and its pdimethyl (DDF), p-nitro (NDF), and p-acetoxy (ADF) analogs in eel Ringer's solution (pH 6, 250C). Half-life (min) kObs/miln Compound
TDF DDF NDF ADF
4.5 )9.0 10 17
X 10-' X 10-5

X 10-3 X 10-3

150 7700 69 41

pared immediately before application to the electroplax, the difference in potency cannot be accounted for by the different half-lives. The striking lack of reactivity of DDF must be due to this compound being predominantly in the paraquinoid resonance form. It was noted previously that the spectrum of DDF resembles that of TDF only in very-strong acid. 18 The observation that NDF is as potent as TDF in inhibiting the carbamylcholine response indicates that the trimethylammonium group of TDF is not contributing tQ the blocking action by this compound, as seemed reasonable to assume in view of its similar structure to phenyltrimethylammonium, a potent depolarizing compound. The diazonium nitrogen has a strong positive charge whose magnitude depends on the para substitution of the phenyl ring. The p-trimethylammonium and the -nitro groups have similarly high electronwithdrawing properties, while the p-acetoxy group is much weaker. Thus, the inhibitory potency of these compounds may be explained by the ease of coupling with the diazonium end. The observations8 that d-tubocurariine and phenyltrimethylammonium protect the binding sites of acetylcholine against the action of TDF were confirmed; these compounds also protect thee binding sites against the action of NDF. Thus, one is led to the conclusion that TDF analogs do indeed interact with the binding sites of the receptor, but that the diazonium group itself, rather than the trimethylammonium group, is implicated in attachment to the receptor bio-

polymer.

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PRoc. N. A. S.

After exposure of the electroplax cell to dithiothreitol, TDF becomes a potent reversible receptor-activator at concentrations 100 times lower than were necessary for the irreversible inhibition of the carbamylcholine response. NDF does not depolarize the membrane after dithiothreitol, but inhibits irreversibly the depolarization induced by TDF. These observations suggest that it is indeed the trimethylammonium group of TDF which specifically induces the depolarization of the reduced receptor. TDF is self-inhibitory; if applied before reduction it no longer causes depolarization. This suggests that either end of the molecule can interact with the same active site on the receptor, but with different affinities. The observation that NDF inhibits acetylcholinesterase about as efficiently as does TDF (Table 2) suggests that, here too, the trimethylammonium group of TDF is not necessary for the reaction with the active site of the enzyme.
TABLE 2. Percentage inhibition of acetyhcholinesterase by TDF, DDF, and NDF.
Concentration (pM)
0.2 TDF
...

DDF
...

NDF 21

0.5 1 2 5 10

20 59 84 92 100

... ...

74 100
...
...

0 0 0

...

We wish to thank Prof. David Nachmansohn and Dr. H. W. Chang for valuable and enjoyable discussions and Dr. Chang for synthesizing ADF. Abbreviations: TDF, p-trimethylammonium benzene diazonium fluoroborate; DDF, its dimethyl analog; NDF, its p-nitro analog; ADF, its p-acetoxy analog. * This work was supported, in part, by a grant from the National Institute for Neurological Diseases NS-07853, from the National Science Foundation grants NSF-GB-6835 and NSF-GB7149, by the National Institutes of Health, grant NS-03304, and by the New York Heart Association Inc. 1 Nachmansohn, D., in Chemical and Molecular Basis of Nerve Activity (New York: Acadeiuc Press, 1959), p. 235. 2 Nachmansohn, D., "Proteins of excitable membranes," Proceedings of the Tenth Annual Basic Science Symposium on Membrane Proteins, Nov. 29-30, 1968, New York Heart Association. J. Gen. Physiol., 54, S187-224 (1965). 3 O'Brien, R. D., and L. P. Gilmour, these PROCEEDINGS, 63, 496 (1969). 4 O'Brien, R. D., L. P. Gilmour, and M. E. Eldefrawi, these PROCEEDINGS, 65, 438 (1970). 6 Takagi, K., M. Ikao, and A. Takahashi, Life Sci., 4, 2165 (1965). 6 Gill, E., and H. P. Rang, Mol. Pharmacol., 2, 284 (1966). 7 Karlin, A., and M. Winnik, these PROCEEDINGS, 60, 668 (1968). 8 Changeux, J.-P., T. R. Podleski, and L. Wofsy, these PROCEEDINGS, 58, 2063 (1967). 9 Wofsy, L., and D. Michaeli, these PROCEEDINGS, 58, 2296 (1967). 10 Fenton, J. W., and S. J. Singer, Biochem. Biophys. Res. Commun., 20, 315 (1965). "1 Traylor, P. S., and S. J. Singer, Biochemistry, 6, 881 (1967). 12 Schiemann, G., and M. Winkelmuller, Chem. Ber., 66B, 727 (1933). 13 Starkey, E. B., in Organic Syntheses, Coil. Vol. II, ed. A. H. Blatt. (New York: John Wiley & Sons, 1943), p. 225. 14 Schoffeniels, E., Biochim. Biophys. Acta, 26, 585 (1957). 16 Higman, H. B., T. R. Podleski, and E. Bartels, Biochim. Biophys. Acta, 75, 187 (1963). 16 Podleski, T. R., J.-C. Meunier, and J.-P. Changeux, these PROCEEDINGS, 63, 1239 (1969). 17 Karlin, A., and E. Bartels, Biochim. Biophys. Ada, 126, 525 (1966). 18 Kazitsyna, L. A., A. V. Knznetsova, 0. A. Korytina, and O. A. Rentov, Dokl. Akad. Vauk., S.S.S.R., 154, 379 (1964); Chem. Abstr., 60, 9177b (1964). 19 March, J., Advanced Organic Chemistry (New York: McGraw-Hill Book Co. 1968).