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Biochimica et Biophysica Acta, 5 4 2 ( 1 9 7 8 ) 1 0 7 - - 1 1 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 28594

A Z I D O P H E N A N T R I D I N I U M COMPOUNDS AS P H O T O A F F I N I T Y LABELS OF CHOLINERGIC PROTEINS *

SIEGFRIED STENGELIN, CHRISTIAN WALTHER and FERDINAND

HUCHO

Fachbereich Biologie der UniversitSt Konstanz, D-7750 Konstanz (G.F.R.) (Received December 28th, 1977)

Summary The synthesis of diazidopropidium and diazidoethidium is described. The applicability of these c o m p o u n d s as photoaffinity labels for cholinergic proteins has been investigated: diazidopropidium inhibits neuromuscular transmission. This inhibition is reversible if the c o m p o u n d is applied in the dark b u t becomes irreversible after irradiation with white light. Inhibition is accompanied by a disappearance of miniature endplate potentials. Electrophysiological analysis of this effect indicates that diazidopropidium acts postsynaptically by blocking the acetylcholine receptors. At the molecular level the action of diazidopropidium and diazidoethidium on acetylcholinesterase has been investigated: both compounds appear to bind to a peripheral acetylcholine binding site of this enzyme. Binding of 125I-labeled a-neurotoxin from Na]a naja siamensis to purified membranes from Torpedo californica electric tissue rich in acetylcholine receptors is diminished after incubation and irradiation with diazidopropidium. A b o u t half of the toxin binding sites appear to be blocked b y the photoaffinity :abel.

Introduction Phgtoaffinity labels are useful tools for investigation of structure and functions of proteins. Their main advantage lies in the possibility of controlling the time point and duration of the reaction. In contrast to normal affinity reagents, the photoaffinity label in the dark can diffuse in an unreactive state to its specific binding site, the reaction being started by irradiation only after equilibrium is reached.

* Some of these experiments have been presented at the Frfihjahrstagung der Deutschen Gesellschaft for Biolo~ische Chemie, Regensburg [14]. Abbreviations: PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate.

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Fig. 1. Phenantridinium azides.

Another advantage is the high reactivity of the photoproduct. The intermediary nitrene (if the label is an azide) or carbene (in the case of diazonium compounds} reacts indiscriminately within the binding site of the label, not requiring special amino acid side-chains or functional groups of the protein. Proteins involved in synaptic transmission at the nicotinic cholinergic synapse, e.g., acetylcholine receptor and acetylcholinesterase, have been investigated by means of a group of photoaffinity labels developed by Kiefer et al. [1]. With one of these labels, 4-azido-2-nitrobenzyltrimethylammoniumfluoroborate, the ligand binding site of the acetylcholine receptor protein complex from Torpedo californica electric tissue has been identified [2]. It has also been shown that the active center and a peripheral ligand binding site of the acetylcholinesterase can be labeled separately with this c o m p o u n d [3]. With the triethyl derivative of this arylazide the axonal triethylammonium binding site, a component of the voltage-dependant potassium channel, has been selectively blocked [4,5]. In this paper we propose a new class of photoaffinity labels for cholinergic proteins, the phenantridinium azides (Fig. 1). Propidium azide is shown to block synaptic transmission probably by reacting with the acetylcholine receptor of the neuromuscular junction. This inhibition becomes irreversible by irradiating the preparation with light in the visible range. Materials and Methods Electrophysiological experiments were performed at room temperature using the M. cutaneus pectoris preparation of the frog Rana esculenta. The preparations were kept in chambers of 2.5 ml volume and were bathed in saline containing (mM): NaC1 115, KC1 2.5, CaC12 1.8; buffered with 5 mM Tris/maleate at pH 6.9--7.0. Synapses were located under the microscope using Nomarski interference optics [6]. Conventional stimulation and recording techniques were used. In some experiments hyperpolarizing constant current pulses were injected via a second intracellular microelectrode at the same rate as that at which the nerve was stimulated, in order to monitor possible changes of the input resistance of a muscle fibre. Drugs were sometimes applied to preparations in which, to avoid twitching of muscle fibres, transmitter release was depressed by the presence of 20 mM MgC12 in the saline. For irradiation of preparations soaked in diazidopropidium (in order to make its action irreversible) a light guide from a 150-W halogen lamp (Schott, Type KL 150 B) was directed onto them from a distance of 3 cm.

109

Synthesis of 3,8-diazido-5-(3-diethylmethylammonium)-propyl-6-phenylphenanthridiniumditetrafluoroborate, (diazidopropidium). All reagents used were of the highest commercially available purity. The reaction was performed in a 100-ml vessel wrapped in aluminium foil. 100 mg (0.144 mmol) propidium iodide (Calbiochem, San Diego, U.S.A.) were dissolved at room temperature in I ml 50% fluoroboric acid (Riedel de Haen, Seeke, G.F.R.). The solution was chilled to --5°C (ice/NaC1) and 1 ml of a solution of 99.2 mg (1.44 mmol) NaNO2 in water was slowly added with stirring. Stirring was continued for 20 min. The reaction mixture was filtered and the product was washed four times with 1 ml diethyl ether to remove the iodine which formed during the reaction. The substance was dried in a vacuum desiccator. Yield: 109 mg. The substance was suspended w i t h o u t further purification or characterization in 20 ml acetone and with stirring and cooling (ice/NaC1) 0.1 ml (0.8 mmol) 50% fluoroboric acid and after this 46.8 mg (0.72 mmol) NaN3 were added. The mixture was stirred for 30 min. All subsequent steps were performed under dim red light because of the sensitivity of the product to light. The solvent was evaporated under vacuum (room temperature). To remove the sodium fluoroborate, 2 ml water were added, briefly shaken and filtered. After washing with an additional 0.5 ml water the substance on the filter was dried overnight under vacuum. Yield: 75.0 mg crude product. For further purification it was dissolved in 0.5 ml acetonitrile and 50 pl 50% fluoroboric acid were added. The solution was chromatographically purified on a column (0.6 × 7.0 cm) filled with Kieselgel 60 (Merck, Darmstadt), particle size 0.063-0.2 mm, with 20 ml acetonitrile as eluant. Solvent of the collected fractions containing the azide was evaporated under vacuum, the residue was redissolved in 0.4 ml acetonitrile and, with stirring on ice, 10 ml ethanol were added very slowly. The precipitate was filtered off, washed with 2 ml ethanol and dried under vacuum. The product was stored at 0°C in the dark. Yield: 56 mg (61%), m.p. 170--175°C (decomposition). The c o m p o u n d was characterized by infrared spectroscopy: No NH-absorption bands were observed between 3.800 and 3.200 cm -1, indicating the quantitative conversion of amino into azide groups. The azide groups were identified by their strong absorption at 2.125 cm -1. Further characterization was obtained by NMR in [2H3]acetonitrile. The spectrum showed a triplet at 1.2 ppm (7.1 Hz, two CH3), a multiplet at 2.1--2.6 ppm (CH2), singlet at 2.83 ppm (CH3), a multiplet at 2.9--3.4 ppm (three CH2), a multiplet at 4.6--5.0 ppm (CH2), a doublet at 7.02 ppm (2.3 Hz, one aromatic H), 7.6--8.1 ppm (multiplet, C6Hs and two aromatic H), a doublet at 8.05 ppm (2.3 Hz, one aromatic H) and a multiplet at 8.9--9.2 ppm (two aromatic H). This spectrum indicated that, besides resplacing two amino for azide groups, the structure of the propidium was unchanged. The ultraviolet spectrum showed absorption maxima at 295 nm (e = 50 640) and at 435 nm (e = 5680).

Synthesis of 3,8-diazido-5-ethyl-6-phenyl-phenantridiniumtetra-fluoroborate (diazidoethidiurn). Diazidoethidium was synthesized following the procedure described for diazidopropidium except for the following details. Because of its low solubility, ethidium bromide was dissolved by suspending 200 mg in 2 ml water and adding 4 ml 50% HBF4 to this mixture. The diazonium salt produced

110 as described above precipitated and was washed with cold ethanol instead of ether. Diazidoethidium, the end product, was purified by crystallization: the crude product was dissolved in a small a m o u n t of acetonitrile, ethanol was added, and crystals formed overnight a t - - 2 0 ° C . Diazidoethidium was characterized by infrared, ultraviolet and NMR spectroscopy. Assay of acetylcholinesterase. Activity was measured according to Ellman et al. [ 7 ], using acetylthiocholine as substrate. Preparation of acetylcholinesterase. 5 g of electric tissue from T. californica frozen in liquid nitrogen were cut into small pieces. After addition of 10 ml of water, the mixture was homogenized at 0°C for 1 min at maximum speed with an Ultra Turrax homogenizer. The homogenate was centrifuged for 30 min at 30 000 ×g. The supernatant contained acetylcholinesterase with a specific activity of 0.4--1.0 mmol/per mg. Preparation of acetylcholine receptor-enriched membranes from T. californica electric tissue was performed as previously described [8]: Electric tissue (60 g) from T. californica was cut into small pieces and homogenized with a Waring blendor for 3 min at maximum speed in 120 ml 0.02 M sodium phosphate buffer, pH 7.4, containing 2 mM EDTA, 0.1 mM PMSF (phenylmethylsulfonylfluoride) and 0.4 M NaC1. This and all subsequent steps were performed at 2°C. The homogenate was centrifuged 90 rain at 27 000 X g and the supernatant was discarded. The pellet was resuspended in 60 ml 0.02 M sodium phosphate buffer, pH 7.4, containing 2 mM EDTA and 0.1 mM PMSF with a Waring blendor at low speed (3 rain). The suspension was centrifuged 90 min at 39 000 X g. The pellet was resuspended and centrifuged again and after this second wash the pellet was suspended in 25 ml of the same buffer. After ! 0 min centrifugation at 1000 × g each 5-ml portion of the supernatant was layered on top of 55 ml of a continuous sucrose gradient (25--50% (w/v) sucrose in distilled water containing 0.02% NAN3). Centrifugation was performed for 6 h at 59 000 × g in a Beckman SW 25 rotor. The preparations revealed upon SDS polyacrylamide gel electrophoresis predominantly the four bands assigned to receptor protein. Specific activity was about 1200 nmol bound ~-toxin/g. Assay of acetylcholine receptor activity. Acetylcholine receptor activity was determined by a Millipore filtration assay [13] with 125I-labeled Naja naja siamensis a-toxin as substrate. 100 tll receptor suspension containing 20 /~g protein were mixed with 50 t~l 3% Triton X-100 (dissolved in Ringer's solution) and 50 tll ~2SI-labeled toxin containing 4 t~g of the toxin dissolved in water. The mixture was incubated for 1 h at room temperature and then diluted with 20 ml Ringer's solution. After 10 min the diluted assay mixture was filtered through a Selectron filter (0.45 t~m pore width). The filter was washed with 10 ml Ringer's solution and radioactivity was determined. The result was corrected for radioactivity obtained from the identical experiment but with 100 t~l water instead of membrane suspension. Results and Discussion The pharmacological action of the phenantridinium compounds was tested on the frog neuromuscular junction. Application of propidium or of diazidopro-

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F i g . 2. E f f e c t o f p r o p i d i u m o n e n d p l a t e p o t e n t i a l a n d i n p u t r e s i s t a n c e i n a f r o g m u s c l e f i b r e . (a) i n j e c t i o n of a 10 nA current pulse (lower trace) causes a hyperpolarization (upper trace) during which the motor n e r v e is s t i m u l a t e d t o e v o k e a n e n d p l a t e p o t e n t i a l . ( b ) A f t e r a p p l i c a t i o n o f 1 m l 1 • 1 0 - 4 M p r o p i d i u m , t h e e n d p l a t e p o t e n t i a l is c o m p l e t e l y b l o c k e d w h e r e a s t h e i n p u t r e s i s t a n c e , as s e e n f r o m t h e a m p l i t u d e o f t h e e l e c t r o t o n i e h y p e r p o l a r i z a t i o n , a n d the m e m b r a n e r e s t i n g p o t e n t i a l (74 m V ) are n o t a f f e c t e d . Transm i t t e r r e l e a s e r e d u c e d b y t h e p r e s e n c e o f 2 0 m M M g 2+. A r r o w i n d i c a t e s s t i m u l a t i o n a r t i f a c t o n b o t h t r a c e s . R e c o r d s are a v e r a g e s o f 3 2 m e a s u r e m e n t s a t a r e p e t i t i o n r a t e o f 1 H z . C a l i b r a t i o n : 5 m V , 5 0 m s .

pidium in the dark led to an instantaneous depression of neuromuscular transmission. Resting membrane potential and input resistance were unaffected at a concentration of 0.1 mM {Fig. 2) and even up to 1 mM which is more than adequate to block endplate potentials completely. Similar results were obtained with a solution of diazidopropidium irradiated with white light prior to the application. The photolytic products had an inhibitory effect on the endplate potentials without influencing the resting potential. The depression or block can be fully reversed by washing the preparation for 3 0 - 6 0 min with normal saline. For diazidopropidium this requires that the preparation be kept in the dark or under red light throughout application and subsequent washing. If, however, a preparation after application of diazidopropidium is exposed to white light the endplate potential amplitude is further

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I F i g . 3. E f f e c t o f d i a z i d o p r o p i d i u m i n t h e d a r k ( 1 ) a n d d u r i n g s u b s e q u e n t e x p o s u r e t o l i g h t (2). E n d p l a t e p o t e n t i a l s are e v o k e d b y s t i m u l a t i o n o f t h e m o t o r n e r v e a t a r a t e o f 0 . 5 Hz. I n o r d e r t o p r e v e n t t h e m u s cle f r o m twitching, the endplate potential amplitude has been reduced by a previous treatment of the p r e p a r a t i o n w i t h 1 m l o f 2 . 5 • 1 0 -4 M d i a z i d o p r o p i d i u m a n d e x p o s u r e t o l i g h t , a n d a s u b s e q u e n t w a s h i n n o r m a l f r o g s a l i n e . A t ( 1 ) d i a z i d o p r o p i d i u m is a p p l i e d a g a i n , 1 m l o f a 6 . 2 5 - 1 0 - s M s o l u t i o n b e i n g injected over the preparation under red light (indicated by mechanical artifacts). This leads to reduction o f e n d p l a t e p o t e n t i a l a m p l i t u d e s w h i c h is p a r t l y r e v e r s e d b y d i f f u s i o n o f t h e d r u g i n t o t h e b a t h . A t ( 2 ) i r r a d i a t i o n w i t h w h i t e l i g h t is s t a r t e d , w h i c h r e s u l t s i n a f u r t h e r r e d u c t i o n o f e n d p l a t e p o t e n t i a l a m p l i t u d e . C a l i b r a t i o n : 2 m V , 3 0 s. A C - c o u p l e d r e c o r d i n g , l o w e r c u t t i n g - o f f f r e q u e n c y : 1 H z .

112 a

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F i g . 4. C o m p a r i s o n o f p r e - a n d p o s t - s y n a p t i c n e u r o m u s c u l a r d e p r e s s i o n . (a) E n d p l a t e p o t e n t i a l r e c o r d e d a t a n e n d p l a t e in a p r e p a r a t i o n w h i c h 15 h p r e v i o u s l y h a d b e e n t r e a t e d b y 1 m l o f 5 ' 1 0 - 4 M d i a z i d o propidium and exposure to light and which subsequently had been washed in normal frog saline and kept at 8°C. Note absence of miniature endplate potentials and the small extent to which endplate potentials fluctuate. (b) Endplate potentials and miniature endplate potentials recorded from an endplate in a prepa r a t i o n w h e r e t r a n s m i t t e r r e l e a s e h a s b e e n r e d u c e d b y l o w e r e d Ca 2+ c o n c e n t r a t i o n ( 0 . 2 m M ) i n t h e s a l i n e a n d t h e p r e s e n c e o f 1 . 8 m M M g 2+. T h e a v e r a g e e n d p l a t e p o t e n t i a l a m p l i t u d e is a p p r o x i m a t e l y t h e s a m e as in (a). Note large fluctuations of endplate potential amplitudes and failure on the 5th trace.

depressed (Fig. 3) and the depression becomes irreversible. After washing off the photolytic products of diazidopropidium from the preparation the endplate potential stays reduced (Fig. 4a) or is absent. Thus, depending on the dose applied, it is possible to achieve permanent depression of any degree. With 5 • 10 -4 M diazidopropidium plus irradiation it was found in several cases that even 15 h of washing was insufficient to reverse the block of transmission. Control preparations which had been subjected to the same treatment but were kept in the dark twitched vigorously on stimulation at that time and showed miniature endplate potentials of normal amplitudes. 12--15 h after treatment with 5 • 10 -4 M diazidopropidium and irradiation with white light the sensitivity of the muscle fibres to directly applied acetylcholine was reduced. This was shown by squirting on 50-pl samples of frog saline containing acetylcholine. Whereas in untreated preparations an acetylcholine concentration of approx. 0.5 mM was just sufficient to cause twitching of some fibres a concentration of approx. 15 mM was required with the photolabeled preparation. The reduction in size or disappearance of endplate potentials was paralleled by a reduction in the size of, or the disappearance of miniature endplate potentials (see Fig. 4). The endplate potential in partially blocked preparations were of high quantal content [9], since their amplitudes hardly fluctuated (Fig. 4a), much less than in normal preparations where the average endplate potential amplitude had been reduced to about the same extent by the presence of Mg 2+ and a reduced level of Ca 2÷ (Fig. 4b). From these and the above observations we conclude that propidium and diazidopropidium have a specific post-synaptic action and exert little, if any effect at the pre-synaptic level. Further proof of the specificity of the new photoaffinity label was obtained on the molecular level. The reversible binding of propidium to acetylcholinesterase has been investigated in detail by Taylor et al. [10,11]. They concluded that it is bound by a peripheral anionic site of this enzyme and they proposed

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its application as a fluorescent probe of this site. We compared the reversible inhibition (i.e. without irradiation) of acetylcholinesterase activity by propidium and diazidopropidium (Fig. 5): At low substrate concentrations b o t h reagents act as non-competitive inhibitors with the substrate acetylthiocholine. At high concentrations the Lineweaver-Burk plots indicate a deviation from this simple inhibition pattern which could be interpreted as an activation of the enzyme. Whatever the mechanism of this enzyme substrat~ interaction may be, the azido derivative shows qualitatively the same effect as propidium itself, but the inhibitory effect is decreased. The azido group appears to lower the affinity of the ligand for its binding site b u t it still binds non-competitively to the peripheral anionic binding site. Because of the non-linearity of the LineweaverBurk plot we cannot quantitate this decrease in terms of K i values. Analogous results were obtained with diazidoethidium. We further investigated the effect of the phenantridinium c o m p o u n d s on purified acetylcholine receptor preparations. Fig. 6 shows that 0.17 mM propidium azide in the dark inhibits only slightly the binding of 12SI-labeled ~-neurotoxin from N . na]a siarnensis venom to receptor-enriched membrane fragments from To c a l c i f o r n i c a electric tissue. Irradiation with white light caused a reduction of toxin binding by a b o u t 60%. This percentage could not be increased by a second incubation and irradiation with the photolabel. The reason for this partial blockage is not clear. One interpretation would be that the propidium azide is not inhibiting competitively all the neurotoxin binding sites of the receptor. This interpretation is supported b y the recent finding [12] that the number of binding sites of the reversible ligand propidium equals one-half of the a-bungarotoxin binding sites. Propidium and diazidopropidium act as cholinergic inhibitors probably because of their quaternary ammonium groups. But we cannot determine the structure of the molecule which is actually incorporated into the protein during irradiation. The reactive intermediate during photolysis can react in many

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ways with itself or with its environment. The thin layer chromatogram of the irradiated photolabel shows many components (as has been seen with other photoaffinity labels before). Furthermore the covalently attached molecule appears to show no fluorescence. Therefore the label cannot be used to introduce a fluorescent reporter group into cholinergic proteins. Instead it may serve for the radioactive labeling of subunits and binding sites and for structural investigations analogous to those described in refs. 2 and 3. The main advantage of the label is its light sensitivity. Low doses of light in the visible range which do not alter or destroy the protein are sufficient to start the reaction. Acknowledgements We thank Mr. Giampiero Bandini for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 138, and the Fonds der Chemischen Industrie. References 1 K i e f e r , H., L i n d s t r o m , J., L e n n o x , E.S. a n d Singer, S.J. ( 1 9 7 0 ) Proc. Natl. A c a d . Sci. U.S. 67, 1 6 8 4 - 1688 2 H u c h o , F., L a y e r , P., Kiefer, H . R . a n d Bandini, G. ( 1 9 7 6 ) Proc. Natl. A c a d . Sci. U.S. 73, 2 6 2 4 - - 2 6 2 8 3 L a y e r , P., Kiefer, H . R . a n d H u c h o , F. ( 1 9 7 6 ) Mol. P h a r m a c o l . 12, 9 5 8 - - 9 6 5 4 H u c h o , F., B e r g m a n , C., D u b o i s , J.M., Rojas, E. and Kiefer, H. ( 1 9 7 6 ) N a t u r e 260, 8 0 2 - - 8 0 4 5 H u c h o , F. ( 1 9 7 7 ) N a t u r e 2 6 7 , 7 1 9 - - 7 2 0 6 D r e y e r , F. a n d P e p e r , K. ( 1 9 7 4 ) Pfliigers A r c h . 348, 2 5 7 - - 2 6 2 7 E l l m a n , G . L . , C o u r t n e y , D.K., A n d r e s , V. a n d F e a t h e r s t o n e , R.M. ( 1 9 6 1 ) B i o c h e m . P h a r m a c o l . 7, 88--95 8 H u c h o , F., Bandini, G. a n d Su~rez-Isla, B.A. ( 1 9 7 8 ) Eur. J. B i o c h e m . 8 3 , 3 3 5 - - 3 4 0 9 Del Castillo, J. a n d K a t z , B. ( 1 9 5 4 ) J. Physiol. L o n d . 124, 5 6 0 - - 5 7 3 10 T a y l o r , P., L w e b u g a - M u k a s a , J., L a p p i , S. a n d R a d e m a c h e r , J, ( 1 9 7 4 ) Mol. P h a r m a c o l . 10, 7 0 3 - - 7 0 8 11 T a y l o r , P. a n d L a p p i , S. ( 1 9 7 5 ) B i o c h e m i s t r y 14, 1 9 8 9 - - 1 9 9 7 12 S a t o r , V., R a f t e r y , M,A. a n d M a r t i n e z - C a t r i o n , M. ( 1 9 7 7 ) Arch. B i o c h e m . B i o p h y s . 184, 9 6 - - 1 0 2 13 M e u n i e r , J.-C., S e a l o c k , R., Olsen, R. a n d C h a n g e u x , J.P. ( 1 9 7 4 ) Eur. J. B i o c h e m . 45, 3 7 1 - - 3 9 4 14 H u c h o , F., Bandini, G., L a y e r , P., S t e n g e l i n , S. a n d Sudrez-Isla, B.A. ( 1 9 7 7 ) H o p p e - S e y l e r ' s Z. Physiol. C h e m . 3 5 8 , 253