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1-Pyrene-Butyrylcholine: a Fluorescent Probe for the Cholinergic

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1-Pyrene-Butyrylcholine: a Fluorescent Probe for the Cholinergic Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3097-3101, August 1975 Biophysics 1-Pyrene-butyrylcholine: A fluorescent probe for the cholinergic system (long-lifetime fluorescent probe/neuromuscular junction/acetylcholine receptor/visualization of synapse) F. J. BARRANTES, B. SAKMANN, R. BONNER, H. EIBL, AND T. M. JOVIN Departments of Molecular Biology, Neurobiology, and Biochemical Kinetics, Max-Planck-Institut fur Biophysikalische Chemie, D-3400 Gottingen-Nikolausberg, West Germany Communicated by Manfred Eigen, May 19,1975 ABSTRACT The action of I-pyrene-butyrylcholine, a probes having intrinsically long excited-state lifetimes along new cholinergic fluorescent probe, as been studied at the with a high sensitivity to environmental factors. With this in cellular level using electrophysiological and fluorescence mind, 1-pyrene-butyrylcholine (PBC) was synthesized, and techniques. The spectroscopic properties of the probe were its action studied at the cellular level under physiological found to be similar to those of pyrene-butyric acid, the ex- cited-state lifetime in air-saturated aqueous solutions being conditions. 92 nsec. At micromolar concentrations the probe was found The fluorescent probe behaves as a nondepolarizing, re- to exert a nondepolarizing, reversible blocking action at the versible blocking agent in a nicotinic synapse, it specifically neuromuscular junction of the frog. The same cholinolytic ef- labels the neuromuscular junction, and it possesses a long ex- fect was observed in hypersensitive denervated muscles. cited-state lifetime. The curare-like cholinergic activity of The synaptic localization of the probe could be observed make it therefore ap- with fluorescence microscopy using sub- and micromolar PBC and its spectroscopic properties concentrations. Treatment of the nerve-muscle preparations propriate for studies of the rotational mobility and intermo- with proteolytic enzymes, resulting in the separation of the lecular interactions of the solubilized and membrane-bound nerve ending from the muscle end-plate, enabled a distinc- AChR and the choline-ester hydrolases, as well as the kinet- tion to be made between the fluorescence arising from these ics of binding to all these macromolecular entities. two parts of the synapse. Intense presynaptic fluorescence was observed, and was not altered by micromolar concentra- tions of a-bungarotoxin, d-tubocurarine, hemicholinium, or MATERIALS AND METHODS cholinesterase inhibitors. Faint reversible staining of the end-plate region was observed in enzymically treated mus- Synthesis of 1-Pyrene-butyrylcholine (PBC). (i) 1-Py- cles and was inhibited by prior treatment with a-bungarotox- rene-butyrylchloride (): A solution of 576 mg (2 mmol) of in. Fluorescent a-toxin revealed similar patterns of fluores- 1-pyrene-butyric acid (Eastman) in 15 ml of dry chloroform cence in the end-plate of enzyme-treated muscles. The post- (refluxed for 1 hr over P205 and distilled) and 1.5 g (12 synaptic localization of the fluorescent probe is therefore tentatively identified as the one producing the cholinolytic mmol) of oxalylchloride were refluxed for 4 hr. Benzene, 45 effect upon binding to acetylcholine receptor sites. ml, was added to the reaction mixture. The solvents were evaporated at 300, together with the excess oxalylchloride. Fluorescence techniques have been widely applied to the The residue, a mixture of I and oxalic acid, was treated with study of biomembranes and model systems (1). In the partic- 10 ml of dry chloroform to dissolve the formed 1-pyrene- ular case of excitable membranes, considerable attention has butyrylchloride. been paid to changes of fluorescence intensity occurring (ii) 1-Pyrene-butyric acid-bromoethyl ester (II): The solu- during nerve conduction (2, 3) or electrical excitation of the tion of I (2 mmol) in chloroform was added dropwise at 200 electroplax (4). In all cases, however, nonspecific fluorescent to a thoroughly stirred mixture of 1 g (8 mmol) of 2-bro- probes have been used, with consequent limitations on the moethanol (freshly distilled), 15 ml of dry chloroform, and 1 biological interpretability of the findings. g (10 mmol) of triethylamine. Stirring of the reaction mix- The introduction of a specific cholinergic fluorescent ture was continued for 1 hr at 200. The solvents were evapo- probe (1-dimethylaminonaphthalene-5-sulfonamidoethyltri- rated at 300, and the residue was dissolved in 30 ml of di-iso- methylammonium perchlorate, DNETMA, ref. 5) has great- propyl ether and treated with 30 ml of 0.5 M HCl to transfer ly contributed to the study of acetylcholine receptor (AChR) traces of triethylamine into the aqueous phase. The ether (6, 7). This dansyl derivative, however, has a short fluores- phase was dried over sodium phosphate and filtered. The fil- cence lifetime (Barrantes, unpublished results) and a rela- trate was evaporated to dryness at 300 and the residue (II) tively complex pharmacological activity (7). Some of the was dissolved in 20 ml of acetone. physico-chemical properties of large macromolecular en- (iii) 1-Pyrene-butyrylcholine (III): The solution of II in tities, such as AChR or acetylcholine hydrolase (EC 3.1.1.7) acetone was mixed with 10 ml of trimethylamine (a 33% so- (AChE), can be more conveniently studied with fluorescent lution in ethanol) and allowed to react at 500 for 12 hr (8). The mixture was evaporated to dryness and the residue was Abbreviations: PBC, 1-pyrene-butyrylcholine (1-pyrene-butyric recrystallized from acetone and dried under reduced pres- acid-choline ester); ACh, acetylcholine; AChR, acetylcholine recep- product, PBC, was dissolved in chlo- tor; AChE, acetylcholine hydrolase (EC 3.1.1.7); BuChE, acylcho- sure. The recrystallized line acyl-hydrolase (EC 3.1.1.8); a-BT, a-bungarotoxin; FITC-a-BT roform/methanol/water (200:15:1 by vol) and chromato- fluorescein isothiocyanate a-bungarotoxin; DNETMA, 1-dimethyl- graphed through a silicic acid column (Silicar CCR 7, Mal- aminonaphthalene-5-sulfonamidoethyltrimethylammonium per- linckrodt) using solvents of increasing polarity. PBC eluted chlorate; m.e.p.ps, miniature endplate potentials. with a 65:15:1 (v/v) mixture, with an overall yield of 65%. 3097 Downloaded by guest on October 4, 2021 3098 Biophysics: Barrantes et al. Proc. Nat. Acad. Sci. USA 72 (1975) c Q25- 0 I 276.5 S/CH3 !1 CH 2(?CH2CH 2CCH2CH 2N-CH3 Br" In- I) 1- 3 z w (I W&A0MOAif-, -Ml %1%44**14- r 342.5 378 -1.0 -z G (t) cIc- ITT "m Ir-ll-"---W"- Alul A w l 0 -ccX) wz cn z 0.15- Il I, I' I1 327 Z, 265 1 III1 pi w IL-) 0. I A I I I 0 0 - 311 F(t) co -0.5 -L w loel 1, I Jo I I1 50 NSEC I 1 311 1 6- CD Q05- w I I -- CD_1- t It106 212 318 424 530 636 74282 8 CHRNNEL 240 260 280 300 320 340 360 380 400 420 440 460 FIG. 1. Structure of 1-pyrene-butyrylcholine bromide (MW 472.4), together with the absorption and fluorescence spectra of a 5.3 MM solution of the probe in 100 mM phosphate buffer (pH 7.0) 0 10 21 3~~18 42 50 3 72 4 (20°). The technical fluorescence emission spectrum ( ) of the same solution is also shown (excitation wavelength, 342.5 nm). The dotted line corresponds to the absorption spectrum. STD]. 0EV I T I ON FIG. 2. Nanosecond fluorescence decay of 5 MM PBC mn air- All steps of the synthesis and the purity of the final deriva- equilibrated frog's Ringer solution. Excitation wavelength: 337 nm. tive were assessed by thin-layer chromatography on silica A Schott KV 360 filter was used on the emission. The upper graph gel using various chloroform/methanol/water mixtures. shows the time histogram of the detected fluorescence photons F(t) and the scattered excitation photons G(t) relative to the tim- C25H3003NBr (PBC monohydrate) ing-pulse of the excitation flash. The smooth curve fitting the ex- Calcd.: C 63.56; H 6.40; N 2.96; Br 16.92 perimental response decay F(t) is the reconstituted convolution of Found: 63.53; 6.54; 2.84; 17.02. the flash and a single exponential fluorescence decay of 92 nsec. The lower graph (in expected sigma) shows the deviation D(t), Interaction of PBC with Choline-esterases. A purified normalized to the expected counting error. The upper insert (A-C) preparation of the specific AChE from Torpedo californica corresponds to the autocorrelation function of the error A(t) = f (kindly provided by Dr. P. Taylor, Univ. of California at San D(T) D(r + t)dr. This function is a sensitive measure of any sys- Diego) and horse serum acylcholine acyl-hydrolase (EC tematic errors~in the fit. In the present case, the fit to the single 3.1.1.8) (BuChE) (Sigma Type IV) were assayed according exponential seems to be appropriate to a high degree of accuracy. to Ellman et al. (9) in the presence of PBC. Spectroscopic Determinations and Data Analysis. Ab- nonoverlap of the thick and thin filaments, as assessed by sorption spectra were recorded with a Cary 16 spectrometer. measurement of the optical diffraction patterns of the mus- Fluorescence spectra were made with a FICA 55 spectroflu- cle fibers using a He-Ne laser. Fluorescence was observed orimeter, which gives quantum-corrected excitation (220- with a Zeiss Universal microscope using incident illumina- 550 nm) and emission (200-800 rm) spectra. Fluorescence tion of the surface muscle fibers with a XBO 150 W/i xenon lifetime determinations were made by the single-photon lamp. A UG5 (Arabs33 nm) and a BG3 (X,na,360 nm) time correlation method with an Ortec 9200 nsec decay ap- Schott filter combination was used for exciting PBC fluores- paratus and a Fabritek 1074 multichannel analyzer, con- cence, and a 418 rim long-pass filter was placed on the emis- nected on-line to a PDP 11-20 computer.
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