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Biochimica et Biophysica Acta, 4 4 4 ( 1 9 7 6 ) 2 5 2 - - 2 6 0 © Elsevier Scientific Publishing Company, Amsterdam
-- P r i n t e d in T h e N e t h e r l a n d s
BBA 27994
STUDIES OF NICOTINIC A C E T Y L C H O L I N E RE CE PT O R PRO T E IN FROM R A T BRAIN
WILLIAM McCHESNEY
MOGRE and ROBERT
N. B R A D Y *
Department o f Biochemistry, Vanderbilt Universily, Nashville, Tenn. 3 7232 (U.S.A.) ( R e c e i v e d F e b r u a r y 3 r d , 197,6)
S u m mar y Specific binding of 12Si.labeled a-bungarotoxin to a 34 800 X g pellet of a whole rat brain hom oge na t e has been obtained at levels of 2 pmol toxin per g of whole brain with a K a of 8 . 1 0 -9 M. Binding is reduced 90% by 10 -s M (+)tubocurarine chloride and 10 -4 M nicotine, whereas concentrations of 10 -4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with 125 I-labeled a-bungarotoxin and soluble acetylcholine receptor protein preparations from Torpedo nobiliana; suggesting that this mammalian r e c e p t o r protein is nicotinic in character. Extraction o f the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ~25I-labeled a-bungarotoxin with a Kd of 5 • 10 -9 M. Nicotine and a-bungarotoxin at concentrations of 10 -s M abolish toxinreceptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex f o r matio n 35--40% at similar concentrations. Eserine sulfate, atropine sulfate, d e c a m e t h o n i u m , and pilocarpine had no effect on complex formation at concentrations of 10 -s M.
Introduction The nicotinic acetylcholine r ecept or protein has been extensively purified from the electric organs of several species and its molecular properties are currently under investigation in numerous laboratories (for review see refs. 1,2). The highly specific a-neurotoxins of elapid snake venoms, particularly a-bungarotoxin, were invaluable tools in these studies, a-Bungarotoxin has also been used to localize and ~uantitate acetylcholine recept or protein in neuromuscular junctions [3--5], cell cultures [6--9] and brain [10,11]. Salvaterra et al. [12] recently examined the subcellular and regional distribution of 12SI-labeled a* To w h o m correspondence should be addressed.
253 bungarotoxin binding in rat brain and concluded that the pattern of toxin binding was consistent with the distribution of specific markers for cholinergic function. We have utilized ~2s I-labeled ~-bungarotoxin to quantitate and pharmacologically characterize the acetylcholine receptor protein of a rat brain particulate fraction. Similar binding studies with solubilized Torpedo receptor protein are reported and the results are compared for the two species. The receptor protein was solubilized from the brain particulate fraction and its pharmacological properties examined. Methods
Preparation of particulate fractions. Torpedo particulate fractions were prepared from the lyophilized electric organs of Torpedo nobiliana [13]. Brain particulate fractions were obtained by decapitating male Sprague Dawley rats (180--220 g) and homogenizing whole brains in a 10 X volume of ice cold 0.32 M sucrose, 0.001 M EDTA, 0.05 M sodium phosphate (pH 7.4). The homogenate was centrifuged at 34 800 X g for 20 min at 0°C. The resulting pellet was either resuspended in 0.05 M sodium phosphate (pH 7.4) at 23°C and used for subsequent studies of particulate bound receptor or used directly to prepare soluble preparations. Extraction of particulate fractions. Receptor protein was extracted from the Torpedo particulate fraction in 0.05 M sodium phosphate, 1% Emulphogene (v/v) as previously reported [ 13]. The soluble rat brain fraction was prepared as follows: The rat brain particulate pellet was (a) resuspended in 10 X vols. of 0.05 M sodium phosphate buffer (pH 7.4), (b) recentrifuged at 34 800 X g for 20 min at 0°C, (c) resuspended in 1% Emulphogene BC-720, 0.05 M sodium phosphate buffer (pH 7.4) followed with stirring for 2 h at 23°C, and (d) recentrifuged at 34 800 X g for 20 min. Approximately 80--90% of the rat brain acetylcholine receptor protein activity was present in the supernatant. 2Si.labele d a-bungarotoxin. Incorporation of ~2s I into purified a-bungarotoxin [13] was achieved by lactoperoxidase catalyzed iodination [14]. The reaction solution was loaded onto an SP Sephadex C-25 column (1.4 X 26 cm) previously equilibrated in 0.01 M sodium phosphate (pH 7.4), and eluted with a linear gradient from 0.01 M sodium phosphate (pH 7.4) to 0.05 M sodium phosphate (pH 7.4), 0.5 M sodium chloride (total volume 500 ml), Two radioactive peaks ]6] were obtained. The major radioactive peak (eluted first on SP Sephadex) was homogenous on the disc gel electrophoresis [15] and migrated simultaneously with native a-bungarotoxin. Further characterization of the protein in this peak demonstrated that 65% of the radioactive label was attributable to diiodotyrosine [6]. Formation of 12 s I-labeled a-bungarotoxin-receptor complex in the presence of excess solubilized Torpedo receptor revealed that 75% of the ~=SI-labeled a-bungar0toxin molecules retained the ability to bind soluble Torpedo receptor protein. The specific radioactivity of ~2SI-labeled a-bungarotoxin was determined to be 32 Ci/mmol. Assay for toxin-receptor protein complex. The extent of complex formation of ~2SI-labeled a-bungarotoxin with soluble Torpedo acetylcholine receptor pro-
254 tein was assayed essentially a c c o r d i n g to the DE-81 anion e x c h a n g e filter disc m e t h o d [ 1 6 ] . Aliquots o f soluble T o r p e d o a c e t y l c h o l i n e r e c e p t o r p r o t e i n were a d d e d to a final v o l u m e o f 1.0 ml of 0.05 M s o d i u m p h o s p h a t e ( p H 7.4), 0.25% E m u l p h o g e n e (v/v). Binding was initiated b y a d d i t i o n o f ~2 s I-labeled c~-bung a r o t o x i n to give a final c o n c e n t r a t i o n o f 2.5 - 10 -s M. A f t e r the r e a c t i o n mixt u r e h a d b e e n allowed to s h a k e for 1 h at 23')C, 5 ml o f 0.01 M s o d i u m phosp h a t e ( p H 7.4) 0.25% E m u l p h o g e n e (v/v) were a d d e d to the s o l u t i o n . T h e solution was t h e n filtered o v e r W h a t m a n DE-81 anion e x c h a n g e discs (previously e q u i l i b r a t e d with the assay buffer). Discs were w a s h e d with 15 ml o f b u f f e r and c o u n t e d in a N u c l e a r Chicago ")'-ray s p e c t r o m e t e r with an e f f i c i e n c y of 67 percent. C o n t r o l s were run b y i n c u b a t i n g s a m p l e s with 9.8 • 10 -6 M native a-bung a r o t o x i n f o r 1 h p r i o r to ~2 s I-labeled t o x i n a d d i t i o n . F o r m a t i o n o f t h e c o m p l e x o f ~2 s I-labeled t o x i n with soluble brain a c e t y l c h o line r e c e p t o r p r o t e i n was d e t e r m i n e d b y a m o d i f i e d a m m o n i u m sulfate precipit a t i o n m e t h o d [ 1 7 ] . A l i q u o t s o f soluble brain e x t r a c t were i n c u b a t e d with shaking at 23 ° in a final v o l u m e o f 2.0 ml o f 0.05 M s o d i u m p h o s p h a t e ( p H 7.4), 1% E m u l p h o g e n e , c o n t a i n i n g 12.6 - 10 -9 M 12Si_labele d a - b u n g a r o t o x i n . At the e n d o f 1 h, 8.0 ml o f 37.5% s a t u r a t e d a m m o n i u m sulfate was a d d e d to each s a m p l e , m i x e d well, and the resulting p r e c i p i t a t e was i m m e d i a t e l y collected b y f i l t r a t i o n t h r o u g h W h a t m a n G F / B glass fiber filter circles. T h e precipitate was w a s h e d with 15 ml o f 30% s a t u r a t e d a m m o n i u m sulfate and radioactivity d e t e r m i n e d b y c o u n t i n g the filter discs. Binding o f l~ Si_labeled a - b u n g a r o t o x i n to p a r t i c u l a t e f r a c t i o n s o f rat brain was d e t e r m i n e d b y s h a k i n g aliquots o f the p a r t i c u l a t e f r a c t i o n (see P r e p a r a t i o n o f Particulate Fractions) with 12 .~I-labeled a - b u n g a r o t o x i n for o n e h o u r at 23 ° C. C o n t r o l s were carried o u t b y t r e a t i n g samples u n d e r identical i n c u b a t i o n conditions with native a - b u n g a r o t o x i n , p r i o r to the a d d i t i o n of ~2Si.labeled toxin. I n c u b a t i o n with ~2 s I-labeled a - b u n g a r o t o x i n was t e r m i n a t e d by c e n t r i f u g a t i o n at 34 8 0 0 × g f o r 20 m i n at 0°C. S u p e r n a t a n t s were discarded and pellets w a s h e d twice b y r e s u s p e n s i o n in 10 ml o f 0.05 M s o d i u m p h o s p h a t e ( p H 7.4). Washed pellets were t h e n r e s u s p e n d e d in 5 ml o f 0.05 M s o d i u m p h o s p h a t e ( p H 7.4) a n d aliquots c o u n t e d to d e t e r m i n e r a d i o a c t i v i t y . Protein was d e t e r m i n e d b y the L o w r y m e t h o d [18] using bovine s e r u m alb u m i n as a s t a n d a r d .
Results B i n d i n g o f L2 Si.labele d a - b u n g a r o t o x i n to rat brain particulate fraction T h e binding o f ~2 s I-labeled a - b u n g a r o t o x i n to a p a r t i c u l a t e f r a c t i o n of whole r a t brain is s h o w n in Fig. 1. The process is s a t u r a b l e a n d m a x i m u m binding occurred in the range o f 4 0 - - 8 0 p m o l 12 s I-labeled a - b u n g a r o t o x i n per g p r o t e i n (2--6 p m o l per g w h o l e brain). H a l f s a t u r a t i o n was r e a c h e d at 8.3 • 10 -9 M toxin. When p a r t i c u l a t e f r a c t i o n s f r o m rat liver ( p r e p a r e d f o l l o w i n g the p r o c e d u r e used for brain) wer~e i n c u b a t e d with 12s I-labeled a - b u n g a r o t o x i n u n d e r conditions identical to t h o s e described for brain, n o specific binding was d e t e c t e d . T h e ability o f various agents to inhibit t o x i n binding is s h o w n in Fig. 2. (+)T u b o c u r a r i n e chloride, a nicotinic a n t a g o n i s t , is a p o t e n t inhibitor, achieving 90% p r o t e c t i o n at 10 -s M. A t r o p i n e sulfate, a p o t e n t m u s c a r i n i c a n t a g o n i s t (inhibits m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r s at 10 -9 M), had little e f f e c t on binding
255 3.5
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£ 2.0 x 1.5
/ ~ % x
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-O.I
I0
2'0
0
i
0.1
I [nM]
3'0 410 50 60 ~.-8UNGAROTOXIN [ n a ]
0.2
70
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0.4
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Fig. 1. B i n d i n g o f 12 S I-labeled c t - b u n g a r o t o x i n to a p a r t i c u l a t e f r a c t i o n o f r a t b r a i n . 5-ml a l i q u o t s o f t h e p a x t i e u l a t e f r a c t i o n (see M e t h o d s ) c o n t a i n i n g 5.8 m g p r o t e i n / r n l ( 0 . 0 8 g o r i g i n a l b r a i n w e i g h t / m l ) w e r e inc u b a t e d in t h e p r e s e n c e o f t h e i n d i c a t e d c o n c e n t r a t i o n s of 1251.1abeled ~ - b u n g a r n t o x i n as d e s c r i b e d in M e t h o d s . C o n t r o l s w e r e r u n f o r e a c h c o n c e n t r a t i o n o f 1251.1abele d c t - b u n g a r o t o x i n by t r e a t i n g s a m p l e s w i t h 2 • 10 -6 M n a t i v e ~ - b u n g a r o t o x i n , p r i o r to a d d i t i o n o f 125 I-labeled c~-bungarotoxin. P o i n t s s h o w n o n the c u r v e r e p r e s e n t t h e d i f f e r e n c e b e t w e e n n o n - p r e t r e a t e d a n d p r e t r e a t e d s a m p l e s . T h e d o u b l e - r e c i p r o c a l p l o t o f t h e d a t a , s h o w n in th," isert, gave a v a l u e of S.3 • 11). 9 M f o r h a l f s a t u r a t i o n .
at c o n c e n t r a t i o n s l o w e r t h a n 10 -4 M. Similarly, eserine sulfate, an a c e t y l c h o linesterase i n h i b i t o r , and choline chloride r e d u c e d binding less t h a n 10% until c o n c e n t r a t i o n s g r e a t e r t h a n 10-4 were present. Salt h a d little e f f e c t o n t o x i n binding even at high c o n c e n t r a t i o n s . Native a - b u n g a r o t o x i n essentially a b o l i s h e d specific t o x i n - a c e t y l c h o l i n e r e c e p t o r p r o t e i n binding at levels as l o w as 10 -8 M. T h e results o f similar b i n d i n g studies w i t h solubilized m e m b r a n e p r o t e i n f r o m e x c i t a b l e m e m b r a n e s o f Torpedo are s h o w n in Fig. 3. T h e i n h i b i t i o n pattern is v e r y similar to t h a t o b t a i n e d with r a t brain particles, suggesting t h a t at least p h a r m a c o l o g i c a l l y the t w o r e c e p t o r s are v e r y similar.
Formation o f brain 12 Si_labele d a-bungarotoxin-receptor protein complex When t h e soluble r a t brain f r a c t i o n was i n c u b a t e d with , s S i . l a b e l e d a - b u n g a r o t o x i n a n d t h e n s u b m i t t e d to gel c h r o m a t o g r a p h y on S e p h a d e x G-100, the e l u t i o n p r o f i l e s h o w n in Fig. 4 was o b t a i n e d . A clean s e p a r a t i o n o f 12 s I-labeled a - b u n g a r o t o x i n - r e c e p t o r p r o t e i n c o m p l e x f r o m '2SI-labeled a - b u n g a r o t o x i n is a c h i e v e d b y this p r o c e d u r e ; the c o m p l e x elutes in the v o i d v o l u m e . T r e a t m e n t o f t h e soluble f r a c t i o n with 1 • 10 -6 M native a - b u n g a r o t o x i n b l o c k e d f o r m a tion o f the ' 2 s I-labeled t o x i n - r e c e p t o r p r o t e i n c o m p l e x ( d o t t e d profile, Fig. 4). As i n d i c a t e d in Fig. 5, f o r m a t i o n o f the soluble ~2 s I-labeled a - b u n g a r o t o x i n a c e t y l c h o l i n e r e c e p t o r p r o t e i n c o m p l e x is s a t u r a b l e and d e m o n s t r a t e s a value f o r h a l f s a t u r a t i o n o f 5 • 10 -9 M ' 2 s I-labeled a - h u n g a r o t o x i n .
256
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- LOG [M] F i g , 2. I n h i b i t i o n o f 1 2 5 1 - l a b e l e d c ~ - b u n g a r o t o x i n b i n d i n g t o r a t b r a i n p a r t i c u l a t e f r a c t i o n s . W h o l e r a t b r a i n p a r t i c u l a t e f r a c t i o n w a s o b t a i n e d as d e s c r i b e d i n M e t h o d s . T e n m l a l i q u o t s o f t h e p a r t i c u l a t e fract i o n c o n t a i n i n g 6.8 m g p r o t e i n / m l (0.09 g original w e i g h t b r a i n / m l ) were s h a k e n for 1 h o u r at 2 3 ' C with v a r y i n g c o n c e n t r a t i o n s o f t h e a b o v e i n d i c a t e d a g e n t s . 1251.1abele d a - b u n g a r o t o x i n was t h e n a d d e d a t a f i n a l c o n c e n t r a t i o n o f 5 • 1 0 - 9 M a n d i n c u b a t i o n a l l o w e d t o c o n t i n u e f o r 1 h. T h e a m o u n t o f b o u n d t o x i n w a s t h e n o b t a i n e d as d e s c r i b e d i n M e t h o d s . C o n t r o l s w e r e i n c u b a t e d w i t h 10 - 6 M n a t i v e a - b u n g a r o t o x i n p r i o r t o a d d i t i o n o f l a b e l e d t o x i n , T h e % i n h i b i t i o n was d e t e r m i n e d b y t h e d e c r e a s e i n c o u n t s c o m pared to nontreated samples.
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- LOG [M] Fig. 3. I n h i b i t i o n o f 125 I - l a b e l e d ~ - b u n g a r o t o x i n b i n d i n g t o a s o l u b i l i z e d a c e t y l e h o l i n c r e c e p t o r p r o t e i n p r e p a r a t i o n f r o m T o r p e d o nobiliana. S a m p l e s c o n t a i n i n g s o l u b l e p r o t e i n a t a c o n c e n t r a t i o n o f 0 . 0 6 m g / m l were i n c u b a t e d w i t h the a b o v e i n d i c a t e d agents at the s t a t e d c o n c e n t r a t i o n s for 1 h at 23~C w i t h shaking. 125 I - l a b e i e d ¢ ~ - b u n g a r o t o x i n w a s t h e n a d d e d t o a f i n a l c o n c e n t r a t i o n o f 2 . 5 - 1 0 -8 M a n d t h e a s s a y perf o r m e d as d e s c r i b e d i n M e t h o d s . T h e % i n h i b i t i o n w a s d e t e r m i n e d b y t h e d e c r e a s e i n c o u n t s c o m p a r e d t o nontreated samples.
257 7; 61
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Fig. 4. C h r o m a t o g r a p h y o f s o l u b i l i z e d b r a i n 125 I - l a b e l e d 0 ~ - b u n g a r o t o x i n - a c e t y l c h o l i n e r e c e p t o r p r o t e i n c o m p l e x o n S e p h a d e x G - 1 0 0 . R a t b r a i n p a r t i c u l a t e f r a c t i o n w a s s o l u b i l i z e d as d e s c r i b e d i n M e t h o d s . 1 0 m l a l i q u o t s o f t h e s o l u b l e f r a c t i o n w e r e i n c u b a t e d f o r I h a t 2 3 ° C i n t h e a b s e n c e o r p r e s e n c e o f 1 0 -6 M n a t i v e ( ~ - b u n g a r o t o x i n . 125 I - l a b e l e d c ~ - b u n g a r o t o x i n w a s t h e n a d d e d a t a f i n a l c o n c e n t r a t i o n o f 5 - 1 0 - 9 M a n d i n c u b a t i o n c o n t i n u e d f o r 1 h. T h e n o n - p r e t r e a t e d ( ) a n d n a t i v e (. . . . . . ) toxin pretreated samples were then chromatographed separately on Sephadex G-100 columns (2.5 X 32 cm). Fractions of 5.0 m l were c o l l e c t e d and r a d i o a c t i v i t y was d e t e r m i n e d .
10
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F i g . 5. F o r m a t i o n o f b r a i n 125 I - l a b e l e d ~ - b u n g a r o t o x i n - a c e t y l c h o l i n e r e c e p t o r p r o t e i n c o m p l e x . A s o l u b l e r a t b r a i n f r a c t i o n w a s p r e p a r e d as d e s c r i b e d i n M e t h o d s . 2 - m l a l i q u o t s o f t h e s o l u b l e f r a c t i o n (6 m g p r o t e i n / m l ) were i n c u b a t e d w i t h s h a k i n g for 1 h at 2 3 ° C in t h e p r e s e n c e of 125i_labele d ~ - b u n g a r o t o x i n at the indicated concentrations. Controls were run for each concentration of labeled toxin by pretreating the s a m p l e s f o r a n a d d i t i o n a l h o u r w i t h 8 2 . 5 gig ( 5 . 2 • 1 0 - 6 M) n a t i v e t ~ - b u n g a r o t o x i n p r i o r t o a d d i t i o n o f labeled toxin. Formation of 125i.labele d ~-bungarotoxin-receptor complex was measured by the ammon i u m s u l f a t e p r e c i p i t a t i o n a s s a y (see M e t h o d s ) . T h e i n s e r t s h o w s t h e d o u b l e r e c i p r o c a l p l o t o f t h e d a t a o b t a i n e d a n d g i v e s a h a l f s a t u r a t i o n c o n c e n t r a t i o n f o r l a b e l e d t o x i n a t 5 . 0 • 1 0 - 9 M.
258 TABLE
1
INHIBITION OF TOXIN COMPLEX
BRAIN ACETYLCHOLINE FORMATION BY VARIOUS
RECEPTOR LIGANDS
PROTEIN-125I-LABELED
c~-BUNGARO-
L i g a n d s w e r e a d d e d t o 2 . 0 - m l a l i q u n t s o f s o l u b l e r a t b r a i n f r a c t i o n ( 6 m g p r o t e i n / m l ) p r e p a r e d as d e s c r i b e d i n M e t h o d s , a n d i n c u b a t e d f o r 1 h w i t h s h a k i n g a t 2 3 C, 1 2 S l . l a b e l e d a - b u n g a r o t o x i n was then a d d e d a t a f i n a l c o n c e n t r a t i o n o f 1 2 . 6 " 1 0 - 9 M a n d i n c u b a t i o n c o n t i n u e d f o r 1 h. L a b e l e d t o x i n - a c c t y l choline receptor protein complex formation was then detcrmincd by the ammonium sulfate precipitation a s s a y ( s e e M e t h o d s ) . T h e % i n h i b i t i o n w a s d e t e r m i n e d b y t h e d e c r e a s e in c o u n t s c o m p a r e d t o t h e n o n treated samples. Ligand
Concentration
(M)
% Inhibition
~-Bungarotoxin
1 O- 5 1.7 • 10 6
100 83
Nicotine Curare Carbachol Decamethnnium Atropine Piloearpinc Eserine Lysozyme NaC1
10-5 10-5 10-5 1 O- 5 10 -5 10-5 10-5 10 -5 10-1
100 35 37 0 0 0 0 0 0
The extent of complex formation is greatly decreased when either (+)-tubocurarine chloride, nicotine, carbachol, or native a-bungarotoxin are included in the assay mixture. As seen in Table I, 10 -S M levels of nicotine and a-bungarotoxin abolish complex formation, whereas, carbachol and d-tubocurarine chloride reduce 12 s I-labeled a-bungarotoxin-complex formation 35-40% at similar concentrations. Decamethonium only reduced complex formation when included in the assay mixture at concentrations greater than 10 -4 M. The neuroactive agent eserine sulfate (an acetylcholinesterase inhibitor), atropine sulfate and pilocarpine, (muscarinic inhibitors) had no effect on complex formation
TABLE
II
EXTRACTION OF PROTEIN COMPLEX
BRAIN
1251.LABELED
~-BUNGAROTOXIN-ACETYLCHOLINE
RECEPTOR
B r a i n p a r t i c u l a t e r e c e p t o r f r a c t i o n s w e r e i n c u b a t e d w i t h 1251_labele d c ~ - b u n g a r o t o x i n as d e s c r i b e d in M e t h o d s . P r o t e c t e d s a m p l e s w e r e i n c u b a t e d w i t h 1 • 1 0 -6 M n a t i v e c ~ - b u n g a r o t o x i n p r i o r t o i n c u b a t i o n with labeled toxin, Non-protected samples were incubated only withlabeled toxin. The resulting washed p e l l e t s c o n t a i n i n g b o u n d 1 2 5 1 . l a b e l e d ~ - b u n g a r o t o x i n w e r e h o m o g e n i z e d in 1 0 m l o f 0 . 0 5 M s o d i u m p h o s p h a t e , p H 7 . 4 c o n t a i n i n g t h e a b o v e d e t e r g e n t s a t t h e i n d i c a t e d c o n c e n t r a t i o n s . T h e s e s o l u t i o n s w e r e inc u b a t e d w i t h s h a k i n g a t 2 3 ° C f o r 2 h a n d t h e n c e n t r i f u g e d a t 3 4 8 0 0 X g, f o r 3 0 r a i n . S u p e r n a t a n t s w e r e removed and pellets were resuspended and c o u n t e d to determine the % counts released. Detergent
% Counts solubilized Non-protected
1% v/v 2%v/vT 1% v/v 2% v / v 1% w/v 2% w / v
T X 100 X 100 Emulphogene Emulphogene Lubrol WX Lubrol WX
BC-720 BC-720
78 81 75 76 78 80
Protected
86 89 72
259 when present at 10 -s M. High levels of salt and lysozyme (a protein of low molecular weight and high pI) have no measurable effect on complex formation. As shown in Table II, several detergents release the brain acetylcholine receptor protein from its membrane matrix. It is important to note that nonspecific binding sites {approximately 15--20% of total binding) are also solubilized and hence are present as contaminants in the solubilized extract. Emulphogene was selected for regular usage because of its low absorbance at 280 nm and its potential for use in spectroscopic studies [ 19]. Discussion
As a-bungarotoxin is used as a tool to characterize and identify the acetylcholine receptor protein in the central nervous system it must be kept in mind that there are distinct differences between the cholinergic systems of the central nervous system and peripheral tissue. Studies in the neuro-anatomical distribution of the main components of the cholinergic system demonstrate that only a fraction of the total neurons of the brain and spinal cord can be cholinergic in nature [20]. Muscarinic and nicotinic types of acetylcholine receptors have been observed in central nervous system neurons, and muscarinic receptors are thought to predominate [21]. Additionally, it should be pointed out that the protein receptor of peripheral tissues resides in either an effector organ or gland, whereas the acetylcholine receptor of the central nervous system is incorporated into neuronal membranes. Because of these added complications only a highly selective affinity label would appear to afford a viable approach to the identification and characterization of the acetylcholine receptor protein in brain. The present study would support previous findings that a-bungarotoxin provides this selectivity [ 10--12]. Our results with iodinated a-bungarotoxin gives toxin binding site concentrations which agree well with those reported by Eterovic and Bennett [10] and Salvaterra et al. [12]. However, we do not observe the loss of activity and specificity encountered by the former investigators [10] when iodine was incorporated into the native toxin. Toxin preparations were used that were up to 60 days old without any significant increase in non-specific binding. No apparent modification of native protein activity was observed. As seen in Figs. 1 and 5, toxin binding is saturable and the concentration of toxin required for half-saturation is similar to that reported by Eterovic and Bennett [ 10]. Striking similarity is observed between the pharmacological profiles for the acetylcholine receptor protein of Torpedo {Fig. 3) and that of brain (Fig. 2, Table I). As reported previously [ 10], the binding definitely demonstrates nicotinic characteristics and extends to brain the observation that a-bungarotoxin binds predominately to nicotinic acetylcholine receptors [22]. This observation supports the previous finding that the acetylcholine receptor protein purified from electric eel, induced an auto immune response when injected into rabbits |231. The successful extraction of the receptor protein from the rat brain particulate fraction with no apparent loss of binding specificity (Table I) is central to the isolation of significant quantities of receptor from brain. The solubilized receptor protein is stable for a period of at least two weeks at 4°C with little or
260
no loss of activity. At the levels of the receptor protein found in brain, only a highly selective affinity chromatography system would offer a feasible approach to receptor isolation, since, at these concentrations purification by classical procedures would seem to be impossible. Affinity chromatography utilizing an a-toxin from elapid snake venom as ligand provides such selectivity and such studies are currently underway in our laboratory. Acknowledgements Appreciation is extended to M. Kate Welch and Robert Oswald for their excellent technical assistance. This study was supported in part by the USPHS Grants N S - 1 1 4 3 9 and MH-08107. R.N.B. is a recipient of an Andrew W. Mellon Foundation Teacher-Scientist Award. References 1 D e R o b e r t i s , E. ( 1 9 7 5 ) S y n a p t i c R e c e p t o r s , pp. 119 -172, Marcel D e k k e r , Inc., New York 2 C o h e n , J.B. and C h a n g c u x , J.P. ( 1 9 7 5 ) A n n u . Rev. P h a r m a c o l . 15, 8 3 - - 1 0 3 3 Berg, D.K., Kelly, R.B., Sargent, P.B., Williarnson, P. and Hall, Z.W. ( 1 9 7 2 ) Prfm. Natl. Acad. Sei. U.S. 69, 1 4 7 - - 1 5 1 4 F'ertuck, H.C. a n d Satpeter, M.M. ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 71, 1 3 7 6 - - 1 3 7 8 5 Hartzell, H.C. and F a m b r o u g h , D.M. ( 1 9 7 3 ) Dee. Biol. 30, 1 5 3 - - 1 6 5 6 Vogel, Z., S y t k o w s k i , A.J. a n d N i r e n b e r g , M.W. ( 1 9 7 2 ) Proc. Natl. Acad. Sci. U.S. 69, 3 1 8 0 - - 3 1 8 4 7 S y t k o w s k i , A.J., Vogel, Z. a n d N i r e n b e r g , M.W. ( 1 9 7 3 ) Proe. Natl. Acad. Sci. U.S. 70, 2 7 0 - 2 7 4 8 G r e e n e , L.A., S y t k o w s k i , A.J,, Vogel, Z. a n d N i r e n b e r g , M.W. ( 1 9 7 3 ) N a t u r e 243, 1 6 3 - - 1 6 6 9 F i s c h b a c h , G.D., H e n k a r t , M.P., Cohen, S.A., Breuer, A.C., W h y s n e r , J. and Neal, F'.M. ( 1 9 7 4 ) in: S y n a p t i c T r a n s m i s s i o n and N e u r o n a l I n t e r a c t i o n ( B e n n e t t , M.V.L., ed.) pp. 259-.-283, R a v e n Press, New Y o r k 10 E t e r o v i c , V.A. a n d B e n n e t t , Ig.L. ( 1 9 7 4 ) Bioehirn. Biophys. Acta 362, 3 4 6 - - 3 5 5 11 S a l v a t e r r a , P.M. and M o o r e , W.J. ( 1 9 7 3 ) B i o e h e m . Biophys. Rcs. C o m r n u n . 55, 1 3 1 1 - - 1 3 1 8 12 S a l v a t e r r a , P.M., Mahler, H . R and Mtmrc, W.J. ( 1 9 7 5 ) J. Biol. C h e m . 250, 6 4 6 9 - - 6 4 7 5 13 Ong, D.E. and B r a d y , R.N. ( 1 9 7 4 ) B i o c h e m i s t r y 13, 2 8 2 2 - - 2 8 2 7 14 Morrison, M. a n d Bayse, G.S. ( 1 9 7 0 ) Bioc.hernistry 9, 2 9 9 5 - - 3 0 0 0 15 Reisfield, R.A., Lewis, U.J. and Williams, D.E. ( 1 9 6 2 ) N a t u r e 195, 2 8 1 - - 2 8 3 16 Schrnidt, J. and R a f t e r y , M.A. ( 1 9 7 3 ) Anal. Biochern. 52, 3 4 9 - - 3 5 4 17 Franklin, G.I. and P o t t e r , L.T. ( 1 9 7 2 ) FEBS L e t t . 28, 1 0 1 - - 1 0 6 . 18 L o w r y , O.H., R o s e b r o u g h , N.J., Farr, A.L. and Randall, R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 265- 275 19 M o o r e , W.M., H o l l a d a y , L.A., P u e t t , D. a n d B r a d y , R.N. ( 1 9 7 4 ) F'EBS Lett. 45, 1 4 5 - - 1 4 9 20 Feldberg, W. a n d Vogt, M. ( 1 9 4 8 ) , J, Physiol. 107, 3 7 2 - - 3 8 1 21 Philis, J.W. ( 1 9 7 0 ) T h e P h a r m a c o l o g y of Synapses, P e r g a m o n Press, New York 22 Lee, C.Y. and Chang, C.C. ( 1 9 6 6 ) Mern. Inst. B u t a n o n . Sao Paulo 33, 555 23 L i n d s t r o m , J. and Patrick, d. ( 1 9 7 4 ) in S y n a p t i e T r a n s m i s s i o n and N e u r o n a l I n t e r a c t i o n ( B e n n e t t , M . V . L . , e d . ) , pp. 191 215, R a v e n Press, New Y o r k
Biochimica et Biophysica Acta, 4 4 4 ( 1 9 7 6 ) 2 5 2 - - 2 6 0 © Elsevier Scientific Publishing Company, Amsterdam
-- P r i n t e d in T h e N e t h e r l a n d s
BBA 27994
STUDIES OF NICOTINIC A C E T Y L C H O L I N E RE CE PT O R PRO T E IN FROM R A T BRAIN
WILLIAM McCHESNEY
MOGRE and ROBERT
N. B R A D Y *
Department o f Biochemistry, Vanderbilt Universily, Nashville, Tenn. 3 7232 (U.S.A.) ( R e c e i v e d F e b r u a r y 3 r d , 197,6)
S u m mar y Specific binding of 12Si.labeled a-bungarotoxin to a 34 800 X g pellet of a whole rat brain hom oge na t e has been obtained at levels of 2 pmol toxin per g of whole brain with a K a of 8 . 1 0 -9 M. Binding is reduced 90% by 10 -s M (+)tubocurarine chloride and 10 -4 M nicotine, whereas concentrations of 10 -4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with 125 I-labeled a-bungarotoxin and soluble acetylcholine receptor protein preparations from Torpedo nobiliana; suggesting that this mammalian r e c e p t o r protein is nicotinic in character. Extraction o f the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds ~25I-labeled a-bungarotoxin with a Kd of 5 • 10 -9 M. Nicotine and a-bungarotoxin at concentrations of 10 -s M abolish toxinreceptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex f o r matio n 35--40% at similar concentrations. Eserine sulfate, atropine sulfate, d e c a m e t h o n i u m , and pilocarpine had no effect on complex formation at concentrations of 10 -s M.
Introduction The nicotinic acetylcholine r ecept or protein has been extensively purified from the electric organs of several species and its molecular properties are currently under investigation in numerous laboratories (for review see refs. 1,2). The highly specific a-neurotoxins of elapid snake venoms, particularly a-bungarotoxin, were invaluable tools in these studies, a-Bungarotoxin has also been used to localize and ~uantitate acetylcholine recept or protein in neuromuscular junctions [3--5], cell cultures [6--9] and brain [10,11]. Salvaterra et al. [12] recently examined the subcellular and regional distribution of 12SI-labeled a* To w h o m correspondence should be addressed.
253 bungarotoxin binding in rat brain and concluded that the pattern of toxin binding was consistent with the distribution of specific markers for cholinergic function. We have utilized ~2s I-labeled ~-bungarotoxin to quantitate and pharmacologically characterize the acetylcholine receptor protein of a rat brain particulate fraction. Similar binding studies with solubilized Torpedo receptor protein are reported and the results are compared for the two species. The receptor protein was solubilized from the brain particulate fraction and its pharmacological properties examined. Methods
Preparation of particulate fractions. Torpedo particulate fractions were prepared from the lyophilized electric organs of Torpedo nobiliana [13]. Brain particulate fractions were obtained by decapitating male Sprague Dawley rats (180--220 g) and homogenizing whole brains in a 10 X volume of ice cold 0.32 M sucrose, 0.001 M EDTA, 0.05 M sodium phosphate (pH 7.4). The homogenate was centrifuged at 34 800 X g for 20 min at 0°C. The resulting pellet was either resuspended in 0.05 M sodium phosphate (pH 7.4) at 23°C and used for subsequent studies of particulate bound receptor or used directly to prepare soluble preparations. Extraction of particulate fractions. Receptor protein was extracted from the Torpedo particulate fraction in 0.05 M sodium phosphate, 1% Emulphogene (v/v) as previously reported [ 13]. The soluble rat brain fraction was prepared as follows: The rat brain particulate pellet was (a) resuspended in 10 X vols. of 0.05 M sodium phosphate buffer (pH 7.4), (b) recentrifuged at 34 800 X g for 20 min at 0°C, (c) resuspended in 1% Emulphogene BC-720, 0.05 M sodium phosphate buffer (pH 7.4) followed with stirring for 2 h at 23°C, and (d) recentrifuged at 34 800 X g for 20 min. Approximately 80--90% of the rat brain acetylcholine receptor protein activity was present in the supernatant. 2Si.labele d a-bungarotoxin. Incorporation of ~2s I into purified a-bungarotoxin [13] was achieved by lactoperoxidase catalyzed iodination [14]. The reaction solution was loaded onto an SP Sephadex C-25 column (1.4 X 26 cm) previously equilibrated in 0.01 M sodium phosphate (pH 7.4), and eluted with a linear gradient from 0.01 M sodium phosphate (pH 7.4) to 0.05 M sodium phosphate (pH 7.4), 0.5 M sodium chloride (total volume 500 ml), Two radioactive peaks ]6] were obtained. The major radioactive peak (eluted first on SP Sephadex) was homogenous on the disc gel electrophoresis [15] and migrated simultaneously with native a-bungarotoxin. Further characterization of the protein in this peak demonstrated that 65% of the radioactive label was attributable to diiodotyrosine [6]. Formation of 12 s I-labeled a-bungarotoxin-receptor complex in the presence of excess solubilized Torpedo receptor revealed that 75% of the ~=SI-labeled a-bungar0toxin molecules retained the ability to bind soluble Torpedo receptor protein. The specific radioactivity of ~2SI-labeled a-bungarotoxin was determined to be 32 Ci/mmol. Assay for toxin-receptor protein complex. The extent of complex formation of ~2SI-labeled a-bungarotoxin with soluble Torpedo acetylcholine receptor pro-
254 tein was assayed essentially a c c o r d i n g to the DE-81 anion e x c h a n g e filter disc m e t h o d [ 1 6 ] . Aliquots o f soluble T o r p e d o a c e t y l c h o l i n e r e c e p t o r p r o t e i n were a d d e d to a final v o l u m e o f 1.0 ml of 0.05 M s o d i u m p h o s p h a t e ( p H 7.4), 0.25% E m u l p h o g e n e (v/v). Binding was initiated b y a d d i t i o n o f ~2 s I-labeled c~-bung a r o t o x i n to give a final c o n c e n t r a t i o n o f 2.5 - 10 -s M. A f t e r the r e a c t i o n mixt u r e h a d b e e n allowed to s h a k e for 1 h at 23')C, 5 ml o f 0.01 M s o d i u m phosp h a t e ( p H 7.4) 0.25% E m u l p h o g e n e (v/v) were a d d e d to the s o l u t i o n . T h e solution was t h e n filtered o v e r W h a t m a n DE-81 anion e x c h a n g e discs (previously e q u i l i b r a t e d with the assay buffer). Discs were w a s h e d with 15 ml o f b u f f e r and c o u n t e d in a N u c l e a r Chicago ")'-ray s p e c t r o m e t e r with an e f f i c i e n c y of 67 percent. C o n t r o l s were run b y i n c u b a t i n g s a m p l e s with 9.8 • 10 -6 M native a-bung a r o t o x i n f o r 1 h p r i o r to ~2 s I-labeled t o x i n a d d i t i o n . F o r m a t i o n o f t h e c o m p l e x o f ~2 s I-labeled t o x i n with soluble brain a c e t y l c h o line r e c e p t o r p r o t e i n was d e t e r m i n e d b y a m o d i f i e d a m m o n i u m sulfate precipit a t i o n m e t h o d [ 1 7 ] . A l i q u o t s o f soluble brain e x t r a c t were i n c u b a t e d with shaking at 23 ° in a final v o l u m e o f 2.0 ml o f 0.05 M s o d i u m p h o s p h a t e ( p H 7.4), 1% E m u l p h o g e n e , c o n t a i n i n g 12.6 - 10 -9 M 12Si_labele d a - b u n g a r o t o x i n . At the e n d o f 1 h, 8.0 ml o f 37.5% s a t u r a t e d a m m o n i u m sulfate was a d d e d to each s a m p l e , m i x e d well, and the resulting p r e c i p i t a t e was i m m e d i a t e l y collected b y f i l t r a t i o n t h r o u g h W h a t m a n G F / B glass fiber filter circles. T h e precipitate was w a s h e d with 15 ml o f 30% s a t u r a t e d a m m o n i u m sulfate and radioactivity d e t e r m i n e d b y c o u n t i n g the filter discs. Binding o f l~ Si_labeled a - b u n g a r o t o x i n to p a r t i c u l a t e f r a c t i o n s o f rat brain was d e t e r m i n e d b y s h a k i n g aliquots o f the p a r t i c u l a t e f r a c t i o n (see P r e p a r a t i o n o f Particulate Fractions) with 12 .~I-labeled a - b u n g a r o t o x i n for o n e h o u r at 23 ° C. C o n t r o l s were carried o u t b y t r e a t i n g samples u n d e r identical i n c u b a t i o n conditions with native a - b u n g a r o t o x i n , p r i o r to the a d d i t i o n of ~2Si.labeled toxin. I n c u b a t i o n with ~2 s I-labeled a - b u n g a r o t o x i n was t e r m i n a t e d by c e n t r i f u g a t i o n at 34 8 0 0 × g f o r 20 m i n at 0°C. S u p e r n a t a n t s were discarded and pellets w a s h e d twice b y r e s u s p e n s i o n in 10 ml o f 0.05 M s o d i u m p h o s p h a t e ( p H 7.4). Washed pellets were t h e n r e s u s p e n d e d in 5 ml o f 0.05 M s o d i u m p h o s p h a t e ( p H 7.4) a n d aliquots c o u n t e d to d e t e r m i n e r a d i o a c t i v i t y . Protein was d e t e r m i n e d b y the L o w r y m e t h o d [18] using bovine s e r u m alb u m i n as a s t a n d a r d .
Results B i n d i n g o f L2 Si.labele d a - b u n g a r o t o x i n to rat brain particulate fraction T h e binding o f ~2 s I-labeled a - b u n g a r o t o x i n to a p a r t i c u l a t e f r a c t i o n of whole r a t brain is s h o w n in Fig. 1. The process is s a t u r a b l e a n d m a x i m u m binding occurred in the range o f 4 0 - - 8 0 p m o l 12 s I-labeled a - b u n g a r o t o x i n per g p r o t e i n (2--6 p m o l per g w h o l e brain). H a l f s a t u r a t i o n was r e a c h e d at 8.3 • 10 -9 M toxin. When p a r t i c u l a t e f r a c t i o n s f r o m rat liver ( p r e p a r e d f o l l o w i n g the p r o c e d u r e used for brain) wer~e i n c u b a t e d with 12s I-labeled a - b u n g a r o t o x i n u n d e r conditions identical to t h o s e described for brain, n o specific binding was d e t e c t e d . T h e ability o f various agents to inhibit t o x i n binding is s h o w n in Fig. 2. (+)T u b o c u r a r i n e chloride, a nicotinic a n t a g o n i s t , is a p o t e n t inhibitor, achieving 90% p r o t e c t i o n at 10 -s M. A t r o p i n e sulfate, a p o t e n t m u s c a r i n i c a n t a g o n i s t (inhibits m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r s at 10 -9 M), had little e f f e c t on binding
255 3.5
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3'0 410 50 60 ~.-8UNGAROTOXIN [ n a ]
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Fig. 1. B i n d i n g o f 12 S I-labeled c t - b u n g a r o t o x i n to a p a r t i c u l a t e f r a c t i o n o f r a t b r a i n . 5-ml a l i q u o t s o f t h e p a x t i e u l a t e f r a c t i o n (see M e t h o d s ) c o n t a i n i n g 5.8 m g p r o t e i n / r n l ( 0 . 0 8 g o r i g i n a l b r a i n w e i g h t / m l ) w e r e inc u b a t e d in t h e p r e s e n c e o f t h e i n d i c a t e d c o n c e n t r a t i o n s of 1251.1abeled ~ - b u n g a r n t o x i n as d e s c r i b e d in M e t h o d s . C o n t r o l s w e r e r u n f o r e a c h c o n c e n t r a t i o n o f 1251.1abele d c t - b u n g a r o t o x i n by t r e a t i n g s a m p l e s w i t h 2 • 10 -6 M n a t i v e ~ - b u n g a r o t o x i n , p r i o r to a d d i t i o n o f 125 I-labeled c~-bungarotoxin. P o i n t s s h o w n o n the c u r v e r e p r e s e n t t h e d i f f e r e n c e b e t w e e n n o n - p r e t r e a t e d a n d p r e t r e a t e d s a m p l e s . T h e d o u b l e - r e c i p r o c a l p l o t o f t h e d a t a , s h o w n in th," isert, gave a v a l u e of S.3 • 11). 9 M f o r h a l f s a t u r a t i o n .
at c o n c e n t r a t i o n s l o w e r t h a n 10 -4 M. Similarly, eserine sulfate, an a c e t y l c h o linesterase i n h i b i t o r , and choline chloride r e d u c e d binding less t h a n 10% until c o n c e n t r a t i o n s g r e a t e r t h a n 10-4 were present. Salt h a d little e f f e c t o n t o x i n binding even at high c o n c e n t r a t i o n s . Native a - b u n g a r o t o x i n essentially a b o l i s h e d specific t o x i n - a c e t y l c h o l i n e r e c e p t o r p r o t e i n binding at levels as l o w as 10 -8 M. T h e results o f similar b i n d i n g studies w i t h solubilized m e m b r a n e p r o t e i n f r o m e x c i t a b l e m e m b r a n e s o f Torpedo are s h o w n in Fig. 3. T h e i n h i b i t i o n pattern is v e r y similar to t h a t o b t a i n e d with r a t brain particles, suggesting t h a t at least p h a r m a c o l o g i c a l l y the t w o r e c e p t o r s are v e r y similar.
Formation o f brain 12 Si_labele d a-bungarotoxin-receptor protein complex When t h e soluble r a t brain f r a c t i o n was i n c u b a t e d with , s S i . l a b e l e d a - b u n g a r o t o x i n a n d t h e n s u b m i t t e d to gel c h r o m a t o g r a p h y on S e p h a d e x G-100, the e l u t i o n p r o f i l e s h o w n in Fig. 4 was o b t a i n e d . A clean s e p a r a t i o n o f 12 s I-labeled a - b u n g a r o t o x i n - r e c e p t o r p r o t e i n c o m p l e x f r o m '2SI-labeled a - b u n g a r o t o x i n is a c h i e v e d b y this p r o c e d u r e ; the c o m p l e x elutes in the v o i d v o l u m e . T r e a t m e n t o f t h e soluble f r a c t i o n with 1 • 10 -6 M native a - b u n g a r o t o x i n b l o c k e d f o r m a tion o f the ' 2 s I-labeled t o x i n - r e c e p t o r p r o t e i n c o m p l e x ( d o t t e d profile, Fig. 4). As i n d i c a t e d in Fig. 5, f o r m a t i o n o f the soluble ~2 s I-labeled a - b u n g a r o t o x i n a c e t y l c h o l i n e r e c e p t o r p r o t e i n c o m p l e x is s a t u r a b l e and d e m o n s t r a t e s a value f o r h a l f s a t u r a t i o n o f 5 • 10 -9 M ' 2 s I-labeled a - h u n g a r o t o x i n .
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I 2
I I
- LOG [M] F i g , 2. I n h i b i t i o n o f 1 2 5 1 - l a b e l e d c ~ - b u n g a r o t o x i n b i n d i n g t o r a t b r a i n p a r t i c u l a t e f r a c t i o n s . W h o l e r a t b r a i n p a r t i c u l a t e f r a c t i o n w a s o b t a i n e d as d e s c r i b e d i n M e t h o d s . T e n m l a l i q u o t s o f t h e p a r t i c u l a t e fract i o n c o n t a i n i n g 6.8 m g p r o t e i n / m l (0.09 g original w e i g h t b r a i n / m l ) were s h a k e n for 1 h o u r at 2 3 ' C with v a r y i n g c o n c e n t r a t i o n s o f t h e a b o v e i n d i c a t e d a g e n t s . 1251.1abele d a - b u n g a r o t o x i n was t h e n a d d e d a t a f i n a l c o n c e n t r a t i o n o f 5 • 1 0 - 9 M a n d i n c u b a t i o n a l l o w e d t o c o n t i n u e f o r 1 h. T h e a m o u n t o f b o u n d t o x i n w a s t h e n o b t a i n e d as d e s c r i b e d i n M e t h o d s . C o n t r o l s w e r e i n c u b a t e d w i t h 10 - 6 M n a t i v e a - b u n g a r o t o x i n p r i o r t o a d d i t i o n o f l a b e l e d t o x i n , T h e % i n h i b i t i o n was d e t e r m i n e d b y t h e d e c r e a s e i n c o u n t s c o m pared to nontreated samples.
CHOLINECHLORIDE_-_-~ o
o
-
40
z
:+)-TUBO CHLORIDE ~ \ 60
ATROPINE ~ \
\
I00 I
I
I
I
7
6
5
4
3
2
I
- LOG [M] Fig. 3. I n h i b i t i o n o f 125 I - l a b e l e d ~ - b u n g a r o t o x i n b i n d i n g t o a s o l u b i l i z e d a c e t y l e h o l i n c r e c e p t o r p r o t e i n p r e p a r a t i o n f r o m T o r p e d o nobiliana. S a m p l e s c o n t a i n i n g s o l u b l e p r o t e i n a t a c o n c e n t r a t i o n o f 0 . 0 6 m g / m l were i n c u b a t e d w i t h the a b o v e i n d i c a t e d agents at the s t a t e d c o n c e n t r a t i o n s for 1 h at 23~C w i t h shaking. 125 I - l a b e i e d ¢ ~ - b u n g a r o t o x i n w a s t h e n a d d e d t o a f i n a l c o n c e n t r a t i o n o f 2 . 5 - 1 0 -8 M a n d t h e a s s a y perf o r m e d as d e s c r i b e d i n M e t h o d s . T h e % i n h i b i t i o n w a s d e t e r m i n e d b y t h e d e c r e a s e i n c o u n t s c o m p a r e d t o nontreated samples.
257 7; 61
1
125I - °(-BUNGAROTOXIN RECEPTOR COMPLEX 0
4
X
-~SI-~(BUNGAROTOXIN
/
u
/ IO
o
15 FRACTION
i
I
I
20
25
30
NUMBER
Fig. 4. C h r o m a t o g r a p h y o f s o l u b i l i z e d b r a i n 125 I - l a b e l e d 0 ~ - b u n g a r o t o x i n - a c e t y l c h o l i n e r e c e p t o r p r o t e i n c o m p l e x o n S e p h a d e x G - 1 0 0 . R a t b r a i n p a r t i c u l a t e f r a c t i o n w a s s o l u b i l i z e d as d e s c r i b e d i n M e t h o d s . 1 0 m l a l i q u o t s o f t h e s o l u b l e f r a c t i o n w e r e i n c u b a t e d f o r I h a t 2 3 ° C i n t h e a b s e n c e o r p r e s e n c e o f 1 0 -6 M n a t i v e ( ~ - b u n g a r o t o x i n . 125 I - l a b e l e d c ~ - b u n g a r o t o x i n w a s t h e n a d d e d a t a f i n a l c o n c e n t r a t i o n o f 5 - 1 0 - 9 M a n d i n c u b a t i o n c o n t i n u e d f o r 1 h. T h e n o n - p r e t r e a t e d ( ) a n d n a t i v e (. . . . . . ) toxin pretreated samples were then chromatographed separately on Sephadex G-100 columns (2.5 X 32 cm). Fractions of 5.0 m l were c o l l e c t e d and r a d i o a c t i v i t y was d e t e r m i n e d .
10
8
~ o /
55
o--
-
2
- 0.2
0
0.2
I
0.4 0.6
0.8
[nM] O
'
'
"
IO
20
50
o~-8U N G A R O T O X I N
)'S
'
IOO [nM]
F i g . 5. F o r m a t i o n o f b r a i n 125 I - l a b e l e d ~ - b u n g a r o t o x i n - a c e t y l c h o l i n e r e c e p t o r p r o t e i n c o m p l e x . A s o l u b l e r a t b r a i n f r a c t i o n w a s p r e p a r e d as d e s c r i b e d i n M e t h o d s . 2 - m l a l i q u o t s o f t h e s o l u b l e f r a c t i o n (6 m g p r o t e i n / m l ) were i n c u b a t e d w i t h s h a k i n g for 1 h at 2 3 ° C in t h e p r e s e n c e of 125i_labele d ~ - b u n g a r o t o x i n at the indicated concentrations. Controls were run for each concentration of labeled toxin by pretreating the s a m p l e s f o r a n a d d i t i o n a l h o u r w i t h 8 2 . 5 gig ( 5 . 2 • 1 0 - 6 M) n a t i v e t ~ - b u n g a r o t o x i n p r i o r t o a d d i t i o n o f labeled toxin. Formation of 125i.labele d ~-bungarotoxin-receptor complex was measured by the ammon i u m s u l f a t e p r e c i p i t a t i o n a s s a y (see M e t h o d s ) . T h e i n s e r t s h o w s t h e d o u b l e r e c i p r o c a l p l o t o f t h e d a t a o b t a i n e d a n d g i v e s a h a l f s a t u r a t i o n c o n c e n t r a t i o n f o r l a b e l e d t o x i n a t 5 . 0 • 1 0 - 9 M.
258 TABLE
1
INHIBITION OF TOXIN COMPLEX
BRAIN ACETYLCHOLINE FORMATION BY VARIOUS
RECEPTOR LIGANDS
PROTEIN-125I-LABELED
c~-BUNGARO-
L i g a n d s w e r e a d d e d t o 2 . 0 - m l a l i q u n t s o f s o l u b l e r a t b r a i n f r a c t i o n ( 6 m g p r o t e i n / m l ) p r e p a r e d as d e s c r i b e d i n M e t h o d s , a n d i n c u b a t e d f o r 1 h w i t h s h a k i n g a t 2 3 C, 1 2 S l . l a b e l e d a - b u n g a r o t o x i n was then a d d e d a t a f i n a l c o n c e n t r a t i o n o f 1 2 . 6 " 1 0 - 9 M a n d i n c u b a t i o n c o n t i n u e d f o r 1 h. L a b e l e d t o x i n - a c c t y l choline receptor protein complex formation was then detcrmincd by the ammonium sulfate precipitation a s s a y ( s e e M e t h o d s ) . T h e % i n h i b i t i o n w a s d e t e r m i n e d b y t h e d e c r e a s e in c o u n t s c o m p a r e d t o t h e n o n treated samples. Ligand
Concentration
(M)
% Inhibition
~-Bungarotoxin
1 O- 5 1.7 • 10 6
100 83
Nicotine Curare Carbachol Decamethnnium Atropine Piloearpinc Eserine Lysozyme NaC1
10-5 10-5 10-5 1 O- 5 10 -5 10-5 10-5 10 -5 10-1
100 35 37 0 0 0 0 0 0
The extent of complex formation is greatly decreased when either (+)-tubocurarine chloride, nicotine, carbachol, or native a-bungarotoxin are included in the assay mixture. As seen in Table I, 10 -S M levels of nicotine and a-bungarotoxin abolish complex formation, whereas, carbachol and d-tubocurarine chloride reduce 12 s I-labeled a-bungarotoxin-complex formation 35-40% at similar concentrations. Decamethonium only reduced complex formation when included in the assay mixture at concentrations greater than 10 -4 M. The neuroactive agent eserine sulfate (an acetylcholinesterase inhibitor), atropine sulfate and pilocarpine, (muscarinic inhibitors) had no effect on complex formation
TABLE
II
EXTRACTION OF PROTEIN COMPLEX
BRAIN
1251.LABELED
~-BUNGAROTOXIN-ACETYLCHOLINE
RECEPTOR
B r a i n p a r t i c u l a t e r e c e p t o r f r a c t i o n s w e r e i n c u b a t e d w i t h 1251_labele d c ~ - b u n g a r o t o x i n as d e s c r i b e d in M e t h o d s . P r o t e c t e d s a m p l e s w e r e i n c u b a t e d w i t h 1 • 1 0 -6 M n a t i v e c ~ - b u n g a r o t o x i n p r i o r t o i n c u b a t i o n with labeled toxin, Non-protected samples were incubated only withlabeled toxin. The resulting washed p e l l e t s c o n t a i n i n g b o u n d 1 2 5 1 . l a b e l e d ~ - b u n g a r o t o x i n w e r e h o m o g e n i z e d in 1 0 m l o f 0 . 0 5 M s o d i u m p h o s p h a t e , p H 7 . 4 c o n t a i n i n g t h e a b o v e d e t e r g e n t s a t t h e i n d i c a t e d c o n c e n t r a t i o n s . T h e s e s o l u t i o n s w e r e inc u b a t e d w i t h s h a k i n g a t 2 3 ° C f o r 2 h a n d t h e n c e n t r i f u g e d a t 3 4 8 0 0 X g, f o r 3 0 r a i n . S u p e r n a t a n t s w e r e removed and pellets were resuspended and c o u n t e d to determine the % counts released. Detergent
% Counts solubilized Non-protected
1% v/v 2%v/vT 1% v/v 2% v / v 1% w/v 2% w / v
T X 100 X 100 Emulphogene Emulphogene Lubrol WX Lubrol WX
BC-720 BC-720
78 81 75 76 78 80
Protected
86 89 72
259 when present at 10 -s M. High levels of salt and lysozyme (a protein of low molecular weight and high pI) have no measurable effect on complex formation. As shown in Table II, several detergents release the brain acetylcholine receptor protein from its membrane matrix. It is important to note that nonspecific binding sites {approximately 15--20% of total binding) are also solubilized and hence are present as contaminants in the solubilized extract. Emulphogene was selected for regular usage because of its low absorbance at 280 nm and its potential for use in spectroscopic studies [ 19]. Discussion
As a-bungarotoxin is used as a tool to characterize and identify the acetylcholine receptor protein in the central nervous system it must be kept in mind that there are distinct differences between the cholinergic systems of the central nervous system and peripheral tissue. Studies in the neuro-anatomical distribution of the main components of the cholinergic system demonstrate that only a fraction of the total neurons of the brain and spinal cord can be cholinergic in nature [20]. Muscarinic and nicotinic types of acetylcholine receptors have been observed in central nervous system neurons, and muscarinic receptors are thought to predominate [21]. Additionally, it should be pointed out that the protein receptor of peripheral tissues resides in either an effector organ or gland, whereas the acetylcholine receptor of the central nervous system is incorporated into neuronal membranes. Because of these added complications only a highly selective affinity label would appear to afford a viable approach to the identification and characterization of the acetylcholine receptor protein in brain. The present study would support previous findings that a-bungarotoxin provides this selectivity [ 10--12]. Our results with iodinated a-bungarotoxin gives toxin binding site concentrations which agree well with those reported by Eterovic and Bennett [10] and Salvaterra et al. [12]. However, we do not observe the loss of activity and specificity encountered by the former investigators [10] when iodine was incorporated into the native toxin. Toxin preparations were used that were up to 60 days old without any significant increase in non-specific binding. No apparent modification of native protein activity was observed. As seen in Figs. 1 and 5, toxin binding is saturable and the concentration of toxin required for half-saturation is similar to that reported by Eterovic and Bennett [ 10]. Striking similarity is observed between the pharmacological profiles for the acetylcholine receptor protein of Torpedo {Fig. 3) and that of brain (Fig. 2, Table I). As reported previously [ 10], the binding definitely demonstrates nicotinic characteristics and extends to brain the observation that a-bungarotoxin binds predominately to nicotinic acetylcholine receptors [22]. This observation supports the previous finding that the acetylcholine receptor protein purified from electric eel, induced an auto immune response when injected into rabbits |231. The successful extraction of the receptor protein from the rat brain particulate fraction with no apparent loss of binding specificity (Table I) is central to the isolation of significant quantities of receptor from brain. The solubilized receptor protein is stable for a period of at least two weeks at 4°C with little or
260
no loss of activity. At the levels of the receptor protein found in brain, only a highly selective affinity chromatography system would offer a feasible approach to receptor isolation, since, at these concentrations purification by classical procedures would seem to be impossible. Affinity chromatography utilizing an a-toxin from elapid snake venom as ligand provides such selectivity and such studies are currently underway in our laboratory. Acknowledgements Appreciation is extended to M. Kate Welch and Robert Oswald for their excellent technical assistance. This study was supported in part by the USPHS Grants N S - 1 1 4 3 9 and MH-08107. R.N.B. is a recipient of an Andrew W. Mellon Foundation Teacher-Scientist Award. References 1 D e R o b e r t i s , E. ( 1 9 7 5 ) S y n a p t i c R e c e p t o r s , pp. 119 -172, Marcel D e k k e r , Inc., New York 2 C o h e n , J.B. and C h a n g c u x , J.P. ( 1 9 7 5 ) A n n u . Rev. P h a r m a c o l . 15, 8 3 - - 1 0 3 3 Berg, D.K., Kelly, R.B., Sargent, P.B., Williarnson, P. and Hall, Z.W. ( 1 9 7 2 ) Prfm. Natl. Acad. Sei. U.S. 69, 1 4 7 - - 1 5 1 4 F'ertuck, H.C. a n d Satpeter, M.M. ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 71, 1 3 7 6 - - 1 3 7 8 5 Hartzell, H.C. and F a m b r o u g h , D.M. ( 1 9 7 3 ) Dee. Biol. 30, 1 5 3 - - 1 6 5 6 Vogel, Z., S y t k o w s k i , A.J. a n d N i r e n b e r g , M.W. ( 1 9 7 2 ) Proc. Natl. Acad. Sci. U.S. 69, 3 1 8 0 - - 3 1 8 4 7 S y t k o w s k i , A.J., Vogel, Z. a n d N i r e n b e r g , M.W. ( 1 9 7 3 ) Proe. Natl. Acad. Sci. U.S. 70, 2 7 0 - 2 7 4 8 G r e e n e , L.A., S y t k o w s k i , A.J,, Vogel, Z. a n d N i r e n b e r g , M.W. ( 1 9 7 3 ) N a t u r e 243, 1 6 3 - - 1 6 6 9 F i s c h b a c h , G.D., H e n k a r t , M.P., Cohen, S.A., Breuer, A.C., W h y s n e r , J. and Neal, F'.M. ( 1 9 7 4 ) in: S y n a p t i c T r a n s m i s s i o n and N e u r o n a l I n t e r a c t i o n ( B e n n e t t , M.V.L., ed.) pp. 259-.-283, R a v e n Press, New Y o r k 10 E t e r o v i c , V.A. a n d B e n n e t t , Ig.L. ( 1 9 7 4 ) Bioehirn. Biophys. Acta 362, 3 4 6 - - 3 5 5 11 S a l v a t e r r a , P.M. and M o o r e , W.J. ( 1 9 7 3 ) B i o e h e m . Biophys. Rcs. C o m r n u n . 55, 1 3 1 1 - - 1 3 1 8 12 S a l v a t e r r a , P.M., Mahler, H . R and Mtmrc, W.J. ( 1 9 7 5 ) J. Biol. C h e m . 250, 6 4 6 9 - - 6 4 7 5 13 Ong, D.E. and B r a d y , R.N. ( 1 9 7 4 ) B i o c h e m i s t r y 13, 2 8 2 2 - - 2 8 2 7 14 Morrison, M. a n d Bayse, G.S. ( 1 9 7 0 ) Bioc.hernistry 9, 2 9 9 5 - - 3 0 0 0 15 Reisfield, R.A., Lewis, U.J. and Williams, D.E. ( 1 9 6 2 ) N a t u r e 195, 2 8 1 - - 2 8 3 16 Schrnidt, J. and R a f t e r y , M.A. ( 1 9 7 3 ) Anal. Biochern. 52, 3 4 9 - - 3 5 4 17 Franklin, G.I. and P o t t e r , L.T. ( 1 9 7 2 ) FEBS L e t t . 28, 1 0 1 - - 1 0 6 . 18 L o w r y , O.H., R o s e b r o u g h , N.J., Farr, A.L. and Randall, R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 265- 275 19 M o o r e , W.M., H o l l a d a y , L.A., P u e t t , D. a n d B r a d y , R.N. ( 1 9 7 4 ) F'EBS Lett. 45, 1 4 5 - - 1 4 9 20 Feldberg, W. a n d Vogt, M. ( 1 9 4 8 ) , J, Physiol. 107, 3 7 2 - - 3 8 1 21 Philis, J.W. ( 1 9 7 0 ) T h e P h a r m a c o l o g y of Synapses, P e r g a m o n Press, New York 22 Lee, C.Y. and Chang, C.C. ( 1 9 6 6 ) Mern. Inst. B u t a n o n . Sao Paulo 33, 555 23 L i n d s t r o m , J. and Patrick, d. ( 1 9 7 4 ) in S y n a p t i e T r a n s m i s s i o n and N e u r o n a l I n t e r a c t i o n ( B e n n e t t , M . V . L . , e d . ) , pp. 191 215, R a v e n Press, New Y o r k
