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Na-channels in membrane fragments from electrophorus electricus electroplaques: Biochemical studies
Neurochemistry International Vol.2, pp.73-80. Pergamon Press Ltd. 1980. Printed in Great Britain.
N a - C H A N N E L S IN M E M B R A N E F R A G M E N T S FROM E L E C T R O P H O R U S E L E C T R I C U S E L E C T R O P L A Q U E S : B I O C H E M I C A L STUDIES
H. H. G r H n h a g e n Institut fHr P h y s i o l o g i s c h e Chemie der U n i v e r s i t ~ t des Saarlandes, D - 6650 Homburg/Saar, F. R. G e r m a n y
ABSTRACT N a - c h a n n e l s are p r e p a r e d in m e m b r a n e fragments from the electric organ of E l e c t r o p h o r u s electricus. To i d e n t i f y N a - c h a n n e l s in b i o c h e m i c a l p r e p a r a t i o n s and to study their binding properties, t e t r o d o t o x i n is t r i t i a t e d to a high specific a c t i v i t y of 18 OOO Ci/mol. This is a c h i e v e d by a defined chemical r o c e d u r e from t e t r o d o t o x i n via a c e t y l a n h y d r o t e t r o d o t o x i n to 3H-tetrodotoxin. H - t e t r o d o t o x i n blocks N a - c u r r e n t in Rana e s c u l e n t a nodes at a c o n c e n t r a t i o n of 3 nM, w h e r e a s its a p p a r e n t d i s s o c i a t i o n constant for binding to E l e c t r o phorus N a - c h a n n e l s is K D = (20 ~ 10) nM. This d i f f e r e n c e may be due to a higher d e n s i t y of negative surface charges in nodal regions. Density gradient m e m b r a n e fractions show specific binding of 3 H - t e t r o d o t o x i n up to 5 pmol/mg. Using c a r r i e r - f r e e column e l e c t r o p h o r e s i s and lectin c h r o m a t o g r a p h y m a x i m a l specific b i n d i n g b e t w e e n 25 and 30 pmol/mg have been obtained. In such m e m b r a n e fractions r e c e p t o r d e n s i t i e s are close to values e x p e c t e d for e x t r a s y n a p t i c regions of the e x c i t a b l e face of the electroplaque. 22Naefflux from v e s i c l e s is c o n t r o l l e d by neurotoxins, when bulk Na- and Kc o n c e n t r a t i o n s are altered at the b e g i n n i n g of an efflux experiment. Efflux data and i n t e r a c t i o n of the v e s i c l e s with lectins suggest a high p r o p o r t i o n of i n s i d e - o u t v e s i c l e s in d e n s i t y g r a d i e n t fractions.
~
KEYWORDS Na-channel; t e t r o d o t o x i n - r e c e p t o r ; t e t r o d o t o x i n tritiation; E l e c t r o p h o r u s e l e c t r i c u s e l e c t r o p l a q u e ; Rana e s c u l e n t a node; t e t r o d o t o x i n - b i n d i n g ; c a r r i e r free e l e c t r o p h o r e s i s ; lectin c h r o m a t o g r a p h y ; 22Na-efflux.
INTRODUCTION The importance of c h e m i c a l m e t h o d s in the field of excitable m e m b r a n e s has been r e c o g n i z e d early by D. N a c h m a n s o h n (1963). The b i o c h e m i c a l analysis of the a c e t y l c h o l i n e r e c e p t o r is an i n s t r u c t i v e example for such investigations: On the basis of e l e c t r i c organs as rich sources of b i o l o g i c a l m a t e r i a l a b r e a k t h r o u g h was a c h i e v e d about ten years ago, when the specific b i n d i n g of a - n e u r o t o x i n s was d i s c o v e r e d (Changeux, Kasai and Lee, 1970; Miledi, M o l i n o f f and Potter, 1971) and a system for functional studies in vitro was d e v e l o p e d (Kasai and Changeux, 1971a, 1971b, 1971c). A f a r - r e a c h i n g p h e n o m e n o l o g i c a l d e s c r i p t i o n of v o l t a g e - s e n s i t i v e N a - c h a n n e l s has been d e v e l o p e d about 30 years ago (Hodgkin and Huxley, 1952; Huxley and St~mpfli, 1949). M o r e recently, e l e c t r o p h y s i o l o g i c a l e v i d e n c e for the gating of N a - p e r m e a b i l i t y was o b t a i n e d (for reviews cf Keynes, 1975, 1979; Armstrong, 1975). Furthermore, m a n y n e u r o t o x i n s have been characterized, i n t e r a c t i n g s p e c i f i c l y w i t h N a - c h a n n e l s in vitro (Narahashi, 1974). The
74
H.H. Gr~inhagen
b ioc h e m i c a l a p p r o a c h to study N a - c h a n n e l s in vitro is based on these e l e c t r o p h y s i o l o g i c a l data. The specific binding of t e t r o d o t o x i n (TTX) and saxitoxin (STX) to N a - c h a n n e l s is used to identify the c o r r e s p o n d i n g receptor in s olu b i l i z e d or m e m b r a n e p r e p a r a t i o n s (Ritehie and Rogart, 1977) and has made p o s s i b l e a c o n s i d e r a b l e degree of p u r i f i c a t i o n (Barnola, V i l l e g a s and Camejo, 1973; Balerna and others, 1975; A g n e w and others, 1978, 1980). The N a - c u r r e n t b l o c k i n g action of these n e u r o t o x i n s and the i n t e r f e r e n c e of other n e u r o t o x i n s like veratridine, b a t r a e h o t o x i n , scorpion toxin and sea anemone toxin with the gating m e c h a n i s m are important tools to study N a - f l u x with b i o c h e m i c a l t e c h n i q u e s (Barnola and Villegas, 1976; V i l l e g a s and others, 1977; Catterall, 1975; de Barry, Fosset and Lazdunski, 1977; Courand, Rochat and Lissitzky, 1980; A b i t a and others, 1977). This c o n t r i b u t i o n d e s c r i b e s an a p p r o a c h finally aiming at a c o r r e l a t i o n of functional N a - c h a n n e l p r o p e r t i e s with b i o c h e m i c a l data. N a - c h a n n e l s are prep a r e d in m e m b r a n e v e s i c l e s from the electric organ of the electric eel E l e c t r o p h o r u s electrieus. TTX is used as a specific tool to identify and to study N a - c h a n n e l s in vitro. For this purpose, a chemical method to tritiate TTX to a high specific activity has been developed. C o n d i t i o n s have been found to study specific Na-efflux. Chemical
Tritiation
of T e t r o d o t o x i n
Early attempts to t r i t i a t e TTX and STX n o n s p e c i f i c l y have only yielded preparations of limited specific a c t i v i t y (Ritchie and Rogart, 1977). More recently a specific exchange p r o c e d u r e to t r i t i a t e STX was found (Ritchie, Rogart and Strichartz, 1976). This p r e p a r a t i o n contains m o l a r amounts of t r i t i u m and turned out to be very useful for b i o c h e m i c a l studies. However, back exchange of t r i t i u m at higher p h y s i o l o g i c a l t e m p e r a t u r e s m a y limit its use. We have used a cyclic chemical pathway, starting with TTX and leading back to 3H-TTX via a c e t y l a n h y d r o - t e t r o d o t o x i n (Gr~nhagen and colleagues, 1980). T r e a t i n g TTX in p y r i d i n e / a c e t i c anhydride leads to an a c e t y l a t i o n and to the formation of an a n h y d r o b r i d g e between C 4 and C 9. After alkaline hydrolysis of the acetyl-groups, the a n h y d r o - l i n k a g e is cleaved in T20/TCI. The formation of the e t h e r - l i n k a g e between C 4 and C 9 is p a r a l l e l e d by an e p i m e r i z a t i o n at C4 (Goto and others, 1965). Since this carbon atom is part of a ring system, the e p i m e r i z a t i o n was expected to e n c o u n t e r c o m p a r a b l y stable hydrogen incorporation. The main a d v a n t a g e of this sequence of known reactions (Tsuda and others, 1964) is the p h y s i o l o g i c a l l y inactive a n h y d r o intermediate. Therefore, chemical c o n d i t i o n s can be o p t i m i z e d in the milligram range using bioassays as an analytical tool. After q u a n t i t a t i v e reaction of initial TTX, an overall yield of ca. I % was o b t a i n e d for recovered TTX. The specific a c t i v i t y of this p r e p a r a t i o n is 18 000 Ci/mol, which is not far from ca. 30 O00 Ci/mol e x p e c t e d for the i n c o r p o r a t i o n of one tritium into one m o l e c u l e of TTX. In the course of months, no back exchange has been obse r v e d in the range of p h y s i o l o g i c a l conditions. Binding
of 3 H - T e t r o d o t o x i n
to N a - C h a n n e l s
For binding studies in vitro, m e m b r a n e fragments from the electric organ of E l e c t r o p h o r u s electricus were p r e p a r e d by d i f f e r e n t i a l and isopycnic d e n s i t y gradient c e n t r i f u g a t i o n (Kasai and Changeux, 1971a). Specific binding being d i s p l a c e d by 10 -6 M reference TTX shows h a l f - s a t u r a t i o n at a c o n c e n t r a t i o n of (20 + 10) nM 3H-TTX. Bioassay with frog nodes reveals an apparent d i s s o c i a t i o n c o n s t a n t of 3 nM in vivo (Gr~nhagen and colleagues, 1980). To prove the higher apparent d i s s o c i a t i o n constant for E l e c t r o p h o r u s e l e c t r i c u s m embr a n e fragments, the c o m p e t i t i o n b e t w e e n 3H-TTX and reference TTX was analyzed. D i s p l a c e m e n t of 3H-TTX by reference TTX is in q u a n t i t a t i v e agreement with the apparent d i s s o c i a t i o n constant of KD = (20 + 10) nM. F u r t h e r more, these e x p e r i m e n t s c o n f i r m e d the c o n c e n t r a t i o n of 3HZTTX as d e t e r m i n e d by bioassay. Thus the apparent d i s s o c i a t i o n c o n s t a n t K D = 3 nM for b l o c k i n g N a - c u r r e n t s in frog nodes in vivo is d e f i n i t e l y much lower than the a p p a r e n t d i s s o c i a t i o n c o n s t a n t for h a l f - s a t u r a t i o n of binding to N a - c h a n n e l s in E l e c t r o p h o r u s electricus m e m b r a n e fragments. This d i f f e r e n c e may be due in part to a high density of negative surface charges in nodal regions (Drouin
Na-Channels in Membrane Fragments
and Neumcke, 1974), w h i c h charged TTX at the node.
increases
the local c o n c e n t r a t i o n
75
of p o s i t i v e l y
To o b t a i n more insight into the m o l e c u l a r basis of TTX binding, the k i n e t i c s of T T X - a s s o c i a t i o n and d i s s o c i a t i o n were measured. Both p r o c e s s e s were o b s e r v e d at the r e s o l u t i o n limit of M i l l i p o r e f i l t r a t i o n techniques. At 0 oc the b i m o l e c u l a r forward rate c o n s t a n t was kf ~ 3 x 105 M - I s e c -I as d e t e r m i n e d from the initial slope (GrHnhagen, Dahl and Reiter, 1980). D i s s o c i a t i o n always showed a fast process with k~ = 2.5 x lO-2sec -1, in some p r e p a r a t i o n s a m i n o r part of T T X b i n d i n g d i s s o c i a t e d more slowly with k6 ~ IO-3sec -I (Fig. I). U s i n g the kinetic c o n s t a n t s o b s e r v e d for the m a j o r part of TTX binding, a t h e r m o d y n a m i c d i s s o c i a t i o n c o n s t a n t of K D ~ 8 x 10 -8 M is calculated, w h i c h is not in c o n t r a d i c t i o n to the value d e r i v e d from c o n c e n t r a t i o n d e p e n d e n t s a t u r a t i o n of binding. I n t e r e s t i n g l y the forward b i n d i n g c o n s t a n t 1.5
TTX • 2OhM kl b = 0,025sec-1(80%)
3000-
kl~ = 8.3 x I0 "/' sec -I (20%} E • LO
2000
erl
o
\
"~"
m
% -0.5
I.ooo
°
~
o
Time [mini
Fig.
I. D i s s o c i a t i o n k i n e t i c s of 3H-TTX. Difference between total b i n d i n g and u n d i s p l a c e d b i n d i n g in the p r e s e n c e of I ~M r e f e r e n c e TTX. M e m b r a n e fragments (i.iO g/ml sucrose density) were i n c u b a t e d w i t h 20 nM 3H-TTX. The full line is c a l c u l a t e d as a s u p e r p o s i t i o n of two d i s s o c i a t i o n p r o c e s s e s with k~ = 2.5 x iO-2sec -I (80 % of the initial amplitude) and k6 = 8.3 x 10 -4 sec -I (20 % of the initial amplitude).
kf is c o n s i d e r a b l y b e l o w a d i f f u s i o n c o n t r o l l e d value. F o r w a r d a s s o c i a t i o n slow c o m p a r e d to in vivo data results also for o t h e r b i o c h e m i c a l preparations, where a p p a r e n t t h e r m o d y n a m i c d i s s o c i a t i o n c o n s t a n t s in the n a n o m o l a r range and k i n e t i c d i s s o c i a t i o n p r o c e s s e s in the m i n u t e time range were found (Krueger and others, 1979, Tab. 3). Identical t h e r m o d y n a m i c d i s s o c i a t i o n c o n s t a n t s in vivo and in vitro have been taken as e v i d e n c e for p r e s e r v e d m o l e c u l a r p r o p e r t i e s of the T T X - r e c e p t o r . If i d e n t i t y results from c o m p e n sation of slowed down forward- and backward-kinetics, this a r g u m e n t is not convincing. A n a l y s i s of the b i n d i n g k i n e t i c s may t h e r e f o r e reveal more insight into this part of the N a - c h a n n e l .
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H.H. Griinhagen
P u r i f i c a t i o n of N a - C h a n n e l Rich Membrane F r a ~ m e n t s If the electric organ of E l e c t r o p h o r u s e l e c t r i c u s is h o m o g e n i z e d by a VirTis, and the low speed supernatant f r a c t i o n a t e d on a sucrose d e n s i t y - g r a d i e n t , the plasma m e m b r a n e markers are e n r i c h e d at d e n s i t i e s between ca. 1.04 and 1.16 g/ml (Kasai and Changeux, 1971a, 1971b, 1971c; Reed and Raftery, 1976; Baumann, C h a n g e u x and Benda, 1970). C o n c e r n i n g s p e c i a l i z a t i o n s of the plasma m e m b r a n e (innervated versus non innervated face, subsynaptic versus extrasynaptic areas, planar regions versus invaginations) there is, however, a c o m p a r a b l y broad d i s t r i b u t i o n of m a r k e r s w i t h i n this range of sucrose densities and an u n a m b i g u o u s c o r r e l a t i o n b e t w e e n d e n s i t y fractions in vitro, and m e m b r a n e sections in vivo is not obvious (Rosenberg and others, 1977). In vivo, N a - c h a n n e l s are likely to be located in e x t r a s y n a p t i c regions of the innervated face. In striated muscle, N a - c h a n n e l s have been functionally detected in transverse tubules (Costatin, 1975), which may well correspond to deep transverse i n v a g i n a t i o n s on the innervated face of the e l e c t r o p l a q u e (Bourgeois and others, 1978). Therefore, our p r e p a r a t i v e efforts aim at a p u r i f i c a t i o n of e x t r a s y n a p t i c m e m b r a n e fragments bearing c h a r a c t e r i s t i c s of the planar or of the i n v a g i n a t e d regions of the innervated face.
TABLE
I
Specific Binding in M e m b r a n e F r a c t i o n s Specific Binding [ p m o l / m ~ 3H-TTX
Density gradient fraction (1.O6 g/ml)
2.6
e-neurotoxin 1.0
after electrophoresis: Zone Zone Zone zone
I II III IV
(cathode)
(anode)
(I 41 28 (I
6.1 22 24 3
Specific binding for s a t u r a t i n g c o n c e n t r a t i o n s of neurotoxins. 3 H - T T X - b i n d i n g is c o r r e c t e d for loss during filtration. Specific binding after electrophoresis represents peak values in I ml fractions.
Using shallow sucrose gradients (Gr~nhagen, Dahl and Reiter, 1980) electrophoretic separations of density g r a d i e n t fractions were obtained with a c a r r i e r - f r e e column technique. T y p i c a l results are shown in Table I. Density gradient fractions in most p r e p a r a t i o n s have specific b i n d i n g of 3H-TTX and of e - n e u r o t o x i n ranging from I to 5 pmol/mg. In the density range of interest (1.O4 to 1.16 g/ml) , e l e c t r o p h o r e s i s yields a f r a c t i o n a t i o n into 4 distinguishable zones. The zone with the highest specific binding of 3H-TTX, i.e. zone III, reveals i n t e r e s t i n g l y a c o m p a r a b l y high specific binding of ~neurotoxin. Qualitatively, c o e x i s t e n c e of the receptors for both types of n e u r o t o x i n s is expected for e x t r a s y n a p t i c regions. Quantitatively, 28 p m o l / m g c o r r e s p o n d to ca. 75 T T X - r e c e p t o r s per square micrometer, if as a first a p p r o x i m a t i o n a m e m b r a n e thickness of 75 ~, a p r o t e i n - t o - l i p i d ratio of 1.5 and a density of I g/ml are assumed. A s s u m i n g furthermore a m o l e c u l a r w e i g h t of 300 OOO for the N a - c h a n n e l and a 1:1 s t o i c h i o m e t r y between T T X - r e c e p t o r and Na-channel, the channel w o u l d r e p r e s e n t 0.8 % of the total protein in these p u r i f i e d m e m b r a n e fragments. In vivo, m o r p h o l o g i c a l analyses have allowed to d e t e r m i n e the density of e - n e u r o t o x i n - r e c e p t o r s in e x t r a s y n a p t i c regions of the innervated face (Bourgeois and others, 1978). A value of 370 ~ 260 um -2 was found for this part of the plasma m e m b r a n e including the invaginations. Using the data for specific binding from Tab. I to estimate the receptor densities, one may conclude, that e l e c t r o p h o r e s i s - z o n e III i) q u a l i t a t i v e l y reveals the c h a r a c t e r i s t i c s of excitable e x t r a s y n a p t i c
Na-Channels in Membrane Fragments
77
m e m b r a n e fragments and appears to r e p r e s e n t ii) q u a n t i t a t i v e l y a p r e p a r a t i o n c o n t a i n i n g 15 to 50 % pure e x t r a s y n a p t i c e x c i t a b l e m e m b r a n e fragments, V e s i c u l a r M o r p h o l o g y of M e m b r a n e F r a g m e n t s and C o n d i t i o n s Ion-Flux
for Specific Na-
Freeze fracture e l e c t r o n m i c r o g r a p h s (prepared by G. Dahl) d e m o n s t r a t e that the m a j o r part of m e m b r a n e fragments in d e n s i t y g r a d i e n t fractions forms v e s i c l e s w i t h d i a m e t e r s from i000 to 5000 ~. W h e n the cells are homogenized, their outer surface may a priori become the outer or the inner surface of a vesicle. Since m e m b r a n e area in i n v a g i n a t i o n s r e p r e s e n t s ca. 90 % of the total e x t r a s y n a p t i c area (Bourgeois and others, 1978) and since these tubules may tend to preserve their inverse o r i e n t a t i o n w h e n homogenized, a c o n s i d e r a b l e p r o p o r t i o n of v e s i c l e s could have an i n s i d e - o u t orientation. To a n a l y z e the o r i e n t a t i o n of v e s i c l e membranes, their b i n d i n g of extrac e l l u l a r l y e x p o s e d g l u c o s i d e - or m a n n o s i d e - m o i e t i e s to s e p h a r o s e - b o u n d conc a n a v a l i n A columns was studied (Edelman, Rutishauser, and Milette, 1971). A f r a c t i o n b r e a k i n g through the column w i t h Ringer e l u t i o n has lower specific b i n d i n g for outer surface m a r k e r s (i.e. 2.6 p m o l / m g for TTX) than the d e n s i t y g r a d i e n t f r a c t i o n a p p l i e d to the column (i.e. 3.6 pmol/mg). P r e s u m a b l y this f r a c t i o n has an e n r i c h e d content of i n s i d e - o u t vesicles. The g l u c o s i d e eluted fraction has highly e n r i c h e d specific b i n d i n g of the outer surface markers TTX (up to 26 pmol/mg) and ~ - n e u r o t o x i n (up to 50 pmol/mg). However, with 6 % it r e p r e s e n t s only a m i n o r p r o p o r t i o n of the r e c o v e r e d material. D e p e n d e n t on the a t t r i b u t i o n of i r r e v e r s i b l y c o l u m n - b o u n d m a t e r i a l (ca. 50 %) to the i n s i d e - o u t or o u t s i d e - o u t population, the i n s i d e - o u t vesicle fraction may be e s t i m a t e d to r e p r e s e n t 40 to 90 % of d e n s i t y g r a d i e n t material. These high values are in a g r e e m e n t w i t h the a b u n d a n c e of t r a n s v e r s e tubules on the i n n e r v a t e d face. A c c o r d i n g l y , i n s i d e - o u t v e s i c l e s could be an e n r i c h e d pop u l a t i o n of m e m b r a n e fragments from t r a n s v e r s e tubules. V e s i c l e s from the e l e c t r i c organ of E l e c t r o p h o r u s e l e c t r i c u s may be used for ion flux studies in vitro. I n v e s t i g a t i o n of specific ion flux through synaptic c h a n n e l s has r e s u l t e d in important c o n t r i b u t i o n s to an unders t a n d i n g of the a c e t y l c h o l i n e - r e c e p t o r (Kasai and Changeux, 1971; Hess, Cash and Aoshima, 1979; Cash and Hess, 1980; Aoshima, Cash and Hess, 1980). In a c o m p a r a b l e way it has been p o s s i b l e to observe N a - f l u x t h r o u g h N a - c h a n n e l s in m i x e d v e s i c l e s from lobster nerve and soybean liposomes, where c h a n n e l s were o p e n e d w i t h v e r a t r i d i n e and b l o c k e d s p e c i f i c l y with t e t r o d o t o x i n (Villegas and coworkers, 1977, 1979). In m e m b r a n e v e s i c l e s from E l e c t r o p h o r u s e l e c t r i c u s e l e c t r i c organ we have no e v i d e n c e for specific effects of N a - c h a n n e l n e u r o t o x i n s on Na-efflux, if v e s i c l e s are e q u i l i b r a t e d inside and outside w i t h Ringer s o l u t i o n (160 mM NaCI, 5 m M KCI, 2 m M CaCI2, 2 m M MgCI2, 1.5 m M p h o s p h a t e buffer, pH 7.0; "Na-Ringer"), a g r a d i e n t e x i s t i n g only for tracer amounts of 22Na. Specific n e u r o t o x i n s were, however, found to control 2 2 N a - e f f l u x from Electrophorus e l e c t r i c u s vesicles, if the v e s i c l e s were e q u i l i b r a t e d before the flux e x p e r i m e n t in N a - R i n g e r and t r a n s f e r r e d at the b e g i n n i n g of the efflux into K - R i n g e r (160 m M KCI, 5 mM NaCI, 2 m M CaCI2, 2 m M MgCI2, 1.5 m M p h o s p h a t e b u f f e r pH 7.0), or vice versa. In such experiments, the o p e n i n g of the Nachannel was p r o l o n g e d by c e v a d i n e (HonerjMger, 1973) or by t r a n s i e n t treatment of i n s i d e - o u t v e s i c l e s w i t h N - b r o m o - a c e t a m i d e (NBA) (Oxford, Wu and Narahashi, 1978). Fig. 2 shows an efflux e x p e r i m e n t after t r e a t m e n t of the v e s i c l e s w i t h NBA. In the p r e s e n c e of TTX, the e f f l u x curve is s i g n i f i c a n t l y shifted to higher amounts of 22Na r e t a i n e d in the v e s i c l e s in this time range. The time r e s o l u t i o n of f i l t r a t i o n t e c h n i q u e s does not a l l o w to m o n i t o r the initial phase of efflux, where the slope of the e f f l u x curve is e x p e c t e d to be steeper in the absence of TTX. An e s t i m a t i o n shows that an e s s e n t i a l part of 22Naefflux may well occur in the m i l l i s e c o n d time range: under conditions as
78
H.H.
Gr{inhagen
NBA, Imin, ImM
OW
MF + TTX o MF x Filters
•
3
o 04 04
"O O
.c_ o O
n.
X~X-...
0
x
i
I
i
I
30
60
90
120
Time [sec] Fig.
2. 2 2 N a - e f f l u x from vesicles (l.ll g/ml sucrose density) t r a n s i e n t l y treated with N - b r o m o a c e t a m i d e (NBA, I min at a c o n c e n t r a t i o n of I mM). Incubation o v e r n i g h t in Na-Ringer, vesicles trapped on a c o m b i n a t i o n of G F / D - p r e f i l t e r and a HAWP M i l l i p o r e - f i l t e r (13 mm), c o n t r o l l e d elution of r el e a s e d tracer with K - R i n g e r (500 ~i per elution step). Mean values from 3 experiments.
chosen for the e x p e r i m e n t in Fig. 2, 160 mM NaCI bulk c o n c e n t r a t i o n and ca. 3 % i n t r a v e s i c u l a r volume y i e l d ca. 5 m M i n t r a v e s i c u l a r Na-ions. A specific T T X - r e c e p t o r c o n c e n t r a t i o n of 2.5 pmol/mg protein and a p r o t e i n c o n c e n t r a t i o n of 2 mg/ml during the e q u i l i b r a t i o n period c o r r e s p o n d to ca. 5 nM Na-channels, if a 1:1 s t o i c h i o m e t r y is assumed for T T X - r e c e p t o r and Na-channels. C o m p a r i n g 5 mM i n t r a v e s i c u l a r N a - i o n s w i t h 5 nM N a - c h a ~ n e l s , a number of 106 Na-ions per channel is obtained. This compares to iO ions passing a single Na-channel per second. The o c c u r r e n c e of specific n e u r o t o x i n effects appears to require d i f f e r i n g Na- and K-ion c o n c e n t r a t i o n s inside and outside the vesicles. One may h y p o t h e s i z e that a m e m b r a n e p o t e n t i a l has to be e s t a b l i s h e d before Nachannels can be e f f i c i e n t l y opened. This c o n d i t i o n may be related to HodgkinHuxley i n a c t i v a t i o n (Hodgkin and Huxley, 1952) or to slower m e c h a n i s m of ina c t i v a t i o n (Brismar, 1977; Neumcke, Schwarz and St~mpfli, 1979). A l t e r n a t i v e ly, asymmetric b i n d i n g of Na- and K-ions to specific sites on o p p o s i t e faces of the m e m b r a n e c o u l d also a c c o u n t for the o b s e r v e d phenomena. The o c c u r r e n c e ofa n e u r o t o x i n effect i n d e p e n d e n t on the d i r e c t i o n of Na- and K - g r a d i e n t s is
Na-Channels in Membrane Fragments
79
probably due to the heterogeneity of the preparation, which contains both outside-out and inside-out vesicles.
CONCLUSIONS Our method to tritiate TTX by a defined chemical procedure yields a preparation of 3H-TTX with a high specific activity. This specific ligand allows us to identify Na-channel-rich membrane fragments in vitro and to study binding kinetics. In density gradient fractions, the specific binding of 3H-TTX is comparable to the specific binding of a-neurotoxins. Correspondingly, this membrane preparation may be as useful for biochemical studies of Na-channels as it has turned out to be for investigations of the acetylcholine-receptor (Kasai and Changeux, 1971a, 1971b, 1971c; Hess, Cash and Aoshima, 1979). Further purification of membrane fragments is possible by carrier-free column electrophoresis. This technique yields fractions with specific binding of TTX and ~-neurotoxin close to values expected for highly purified extrasynaptic fragments of the excitable face of the electroplaque (Bourgeois and colleagues, 1978). Such a preparation is of interest for spectroscopic studies, where high densities of receptors are of major importance. The vesicular morphology of most membrane fragments is of high interest for functional studies in vitro. A high proportion of vesicles in density gradient fractions is inside-out. Although this heterogeneity may complicate a quantitative analysis of ion flux data, after purification outside-out and insideout fractions of vesicles will certainly be useful for studies of vectorial phenomena and sidedness. Specific effects of neurotoxins on ion flux were observed, when Na- and Kion concentrations were modified between equilibration with the tracer and start of an efflux experiment. Further experiments have to analyze the molecular basis of this phenomenon to allow us to correlate molecular and functional data.
ACKNOWLEDGEMENT Part of the data have been obtained in cooperation with Drs. H. Fasold, G. Dahl, M. Rack and R. St~mpfli. The support of this work by V. Ullrich and R. St~mpfli and the expert assistance of W. BHhler, P. Gries, P. Reiter and R. Stolz are gratefully acknowledged. Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 38 "Membranforschung".
REFERENCES Abita, J.P., R. Chicheportiche, H. Schweitz, and M. Lazdunski (1977). Biochemistry, 16, 1838-1844. Agnew, W.S., S. R. Levinson, J. S. Brabson, and M. A. Raftery (1978). Proc.Natl.Acad. Sci.USA, 75, 2606-2610. Agnew, W. S., A. C. Moore, S. R. Levinson, and M. A. Raftery (1980). Biochem. Biophys. Res.Comm., 92, 860-866. Aoshima, H., D. J. Cash, and G. P. Hess (1980). Biochem. Biophys.Res.Comm., 9_22, 896-904. Armstrong, C. M. (1975). Quart. Rev.Biophys.,[, 179-210. Balerna, M., M. Fosset, R. Chicheportiche, G. Romey, and M. Lazdunski (1975). Biochemistry , i_44, 55OO-5511. Barnola, F. V., R. Villegas, and G. Camejo (1973). Biochim. Biophys.Acta, 298, 84-94. Barnola, F. V., and R. Villegas (1976). J.Gen. Physiol., 67, 81-90. de Barry, J., M. Fosset, and M. Lazdunski (1977). Biochemistry, 16, 38503855.
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Bauman, A., J. P. Changeux, P. Benda (1970). FEBS Lett., 8, 145-148. Bourgeois, J. P., J. L. Popot, A° Ryter, and J. P. C h a n g e u x (1978). J. Cell Biology, 79, 2OO-216. Brismar, T. (1977). J. Physiol. London, 270, 283-297. Catterall, W. A. (1975). Proc.Natl.Acad. Sci.USA, 72, 1782-1786. Cash, D. J., and G. P. Hess (1980). Proc.Natl.Acad. Sci.USA, 77, 842-846. Changeux, J.P., M. Kasai, and C. Y. Lee (1970). Proc.Natl.Acad. Sci.USA, 67, 1241-1247. Costatin, L. L. (1975). F e d e r a t i o n Proc., 34, 1390-1394. Couraud, F., H. Rochat, and S. Lissitzky (1980). Biochemistry, 19, 457-462. Drouin, H., and B. Neumcke (1974). Pfl~gers Arch., Eur.J.Physiol., 351, 207-229. Edelman, G. M., U. Rutishauser, and C. F. Milette (1971). Proc.Natl.Acad. Sci.USA, 68, 2153-2157. Goto, T., Y. Kishi, S. Takahashi, and Y. Hirata (1965). Tetrahedron, 21, 2059-2088. Gr~nhagen, H. H., M. Rack, R. St~hapfli, H. Fasold, and P. Reiter (submitted for publication). Gr~nhagen, H. H., G. Dahl, and P. Reiter (1980). In preparation. Gr~nhagen, H. H. (1980). In preparation. Hess, G. P., D. J. Cash, and H. A o s h i m a (1979). Nature, 282, 329-331. Hodgkin, A. L., and A. F. Huxley (1952). J. Physiol. (London), 117, 500-544. Honerj~ger, P. (1973). N a u n y n - S c h m i e d e b e r g ' s Arch. Pharmacol.,280, 391-416. Huxley, A. F., and R. St~mpfli (1949). J. Physiol. (London), 108, 315-339. Kasai, M., and J. P. C h a n g e u x (1971). J. Membr. Biol., 6, 1-23. Kasai, M., and J. P. C h a n g e u x (1971). J. Membr. Biol., 6, 24-57. Kasai, M., and J. P. C h a n g e u x (1971). J. Membr. Biol., ~, 58-80. Keynes, R. D. (1975). In The Nervous System, D. B. Tower, Ed., Vol. I: The Basic Neurosciences, pp. 165-175, Raven Press, New York. Keynes, R. D. (1979). Sci. Amer., 240, 98-107. Krueger, B. K., R. W. Ratzlaff, G. R. Strichartz, and M. P. Blaustein (1979). J. Membr. Biol.,50, 287-310. Miledi, R., P. Molinoff, and L. Potter (1971). Nature, 229, 554-557. Nachmansohn, D. (1963). In Handbuch der e x p e r i m e n t e l l e n Pharmakolo@ie, Erg ~ n z u n g s w e r k XV (0. Eichler, A. Farah, Eds.) pp. 701-740, Springer, Berlin. Narahashi, T. (1974). Phys. Rev., 54, 813-889. Neumcke, B., W. Schwarz, and R. St~mpfli (1979). Biochim. Biophys.Acta, 558, 113-118. Oxford, G. S., C. H. Wu, and T. N a r a h a s h i (1978). J. Gen. Physiol., 71, 227-247. Reed, J. K., and M. A. Raftery (1976). Biochemistry, 15, 944-953. Ritchie, J. M., R. B. Rogart, and G. Strichartz (1976). J. Physiol. (London), 261, 477-494. Ritchie, J. M., and R. B. Rogart (1977). Rev.Physiol. Biochem. Pharmacol., 79, 1-50. Rosenberg, P., I. Silman, E. Ben-David, A. DeVries, and E. Condrea (1977). J. Neurochemistry, 29, 561-578. Tsuda, K., S. Ikuma, M. Kawamura, R. Tachikawa, K. Sakai, C. Tamura, and O. A m a k a s u (1964). Chem. Pharm. Bull., i_22, 1357-1374. Villegas, R., G. M. Villegas, F. V. Barnola, and E. Racker (1977). Biochem. Biophys. Res. Comm., 79, 210-217. Villegas, R., G. M. Villegas, F. V. Barnola, and E. Racker (1979). In Advances in C y t o p h a r m a c o l o ~ y (B. Ceccarelli, F. Clementi, Eds.) Vol. 3, pp. 373-385, Raven Press, New York.
N a - C H A N N E L S IN M E M B R A N E F R A G M E N T S FROM E L E C T R O P H O R U S E L E C T R I C U S E L E C T R O P L A Q U E S : B I O C H E M I C A L STUDIES
H. H. G r H n h a g e n Institut fHr P h y s i o l o g i s c h e Chemie der U n i v e r s i t ~ t des Saarlandes, D - 6650 Homburg/Saar, F. R. G e r m a n y
ABSTRACT N a - c h a n n e l s are p r e p a r e d in m e m b r a n e fragments from the electric organ of E l e c t r o p h o r u s electricus. To i d e n t i f y N a - c h a n n e l s in b i o c h e m i c a l p r e p a r a t i o n s and to study their binding properties, t e t r o d o t o x i n is t r i t i a t e d to a high specific a c t i v i t y of 18 OOO Ci/mol. This is a c h i e v e d by a defined chemical r o c e d u r e from t e t r o d o t o x i n via a c e t y l a n h y d r o t e t r o d o t o x i n to 3H-tetrodotoxin. H - t e t r o d o t o x i n blocks N a - c u r r e n t in Rana e s c u l e n t a nodes at a c o n c e n t r a t i o n of 3 nM, w h e r e a s its a p p a r e n t d i s s o c i a t i o n constant for binding to E l e c t r o phorus N a - c h a n n e l s is K D = (20 ~ 10) nM. This d i f f e r e n c e may be due to a higher d e n s i t y of negative surface charges in nodal regions. Density gradient m e m b r a n e fractions show specific binding of 3 H - t e t r o d o t o x i n up to 5 pmol/mg. Using c a r r i e r - f r e e column e l e c t r o p h o r e s i s and lectin c h r o m a t o g r a p h y m a x i m a l specific b i n d i n g b e t w e e n 25 and 30 pmol/mg have been obtained. In such m e m b r a n e fractions r e c e p t o r d e n s i t i e s are close to values e x p e c t e d for e x t r a s y n a p t i c regions of the e x c i t a b l e face of the electroplaque. 22Naefflux from v e s i c l e s is c o n t r o l l e d by neurotoxins, when bulk Na- and Kc o n c e n t r a t i o n s are altered at the b e g i n n i n g of an efflux experiment. Efflux data and i n t e r a c t i o n of the v e s i c l e s with lectins suggest a high p r o p o r t i o n of i n s i d e - o u t v e s i c l e s in d e n s i t y g r a d i e n t fractions.
~
KEYWORDS Na-channel; t e t r o d o t o x i n - r e c e p t o r ; t e t r o d o t o x i n tritiation; E l e c t r o p h o r u s e l e c t r i c u s e l e c t r o p l a q u e ; Rana e s c u l e n t a node; t e t r o d o t o x i n - b i n d i n g ; c a r r i e r free e l e c t r o p h o r e s i s ; lectin c h r o m a t o g r a p h y ; 22Na-efflux.
INTRODUCTION The importance of c h e m i c a l m e t h o d s in the field of excitable m e m b r a n e s has been r e c o g n i z e d early by D. N a c h m a n s o h n (1963). The b i o c h e m i c a l analysis of the a c e t y l c h o l i n e r e c e p t o r is an i n s t r u c t i v e example for such investigations: On the basis of e l e c t r i c organs as rich sources of b i o l o g i c a l m a t e r i a l a b r e a k t h r o u g h was a c h i e v e d about ten years ago, when the specific b i n d i n g of a - n e u r o t o x i n s was d i s c o v e r e d (Changeux, Kasai and Lee, 1970; Miledi, M o l i n o f f and Potter, 1971) and a system for functional studies in vitro was d e v e l o p e d (Kasai and Changeux, 1971a, 1971b, 1971c). A f a r - r e a c h i n g p h e n o m e n o l o g i c a l d e s c r i p t i o n of v o l t a g e - s e n s i t i v e N a - c h a n n e l s has been d e v e l o p e d about 30 years ago (Hodgkin and Huxley, 1952; Huxley and St~mpfli, 1949). M o r e recently, e l e c t r o p h y s i o l o g i c a l e v i d e n c e for the gating of N a - p e r m e a b i l i t y was o b t a i n e d (for reviews cf Keynes, 1975, 1979; Armstrong, 1975). Furthermore, m a n y n e u r o t o x i n s have been characterized, i n t e r a c t i n g s p e c i f i c l y w i t h N a - c h a n n e l s in vitro (Narahashi, 1974). The
74
H.H. Gr~inhagen
b ioc h e m i c a l a p p r o a c h to study N a - c h a n n e l s in vitro is based on these e l e c t r o p h y s i o l o g i c a l data. The specific binding of t e t r o d o t o x i n (TTX) and saxitoxin (STX) to N a - c h a n n e l s is used to identify the c o r r e s p o n d i n g receptor in s olu b i l i z e d or m e m b r a n e p r e p a r a t i o n s (Ritehie and Rogart, 1977) and has made p o s s i b l e a c o n s i d e r a b l e degree of p u r i f i c a t i o n (Barnola, V i l l e g a s and Camejo, 1973; Balerna and others, 1975; A g n e w and others, 1978, 1980). The N a - c u r r e n t b l o c k i n g action of these n e u r o t o x i n s and the i n t e r f e r e n c e of other n e u r o t o x i n s like veratridine, b a t r a e h o t o x i n , scorpion toxin and sea anemone toxin with the gating m e c h a n i s m are important tools to study N a - f l u x with b i o c h e m i c a l t e c h n i q u e s (Barnola and Villegas, 1976; V i l l e g a s and others, 1977; Catterall, 1975; de Barry, Fosset and Lazdunski, 1977; Courand, Rochat and Lissitzky, 1980; A b i t a and others, 1977). This c o n t r i b u t i o n d e s c r i b e s an a p p r o a c h finally aiming at a c o r r e l a t i o n of functional N a - c h a n n e l p r o p e r t i e s with b i o c h e m i c a l data. N a - c h a n n e l s are prep a r e d in m e m b r a n e v e s i c l e s from the electric organ of the electric eel E l e c t r o p h o r u s electrieus. TTX is used as a specific tool to identify and to study N a - c h a n n e l s in vitro. For this purpose, a chemical method to tritiate TTX to a high specific activity has been developed. C o n d i t i o n s have been found to study specific Na-efflux. Chemical
Tritiation
of T e t r o d o t o x i n
Early attempts to t r i t i a t e TTX and STX n o n s p e c i f i c l y have only yielded preparations of limited specific a c t i v i t y (Ritchie and Rogart, 1977). More recently a specific exchange p r o c e d u r e to t r i t i a t e STX was found (Ritchie, Rogart and Strichartz, 1976). This p r e p a r a t i o n contains m o l a r amounts of t r i t i u m and turned out to be very useful for b i o c h e m i c a l studies. However, back exchange of t r i t i u m at higher p h y s i o l o g i c a l t e m p e r a t u r e s m a y limit its use. We have used a cyclic chemical pathway, starting with TTX and leading back to 3H-TTX via a c e t y l a n h y d r o - t e t r o d o t o x i n (Gr~nhagen and colleagues, 1980). T r e a t i n g TTX in p y r i d i n e / a c e t i c anhydride leads to an a c e t y l a t i o n and to the formation of an a n h y d r o b r i d g e between C 4 and C 9. After alkaline hydrolysis of the acetyl-groups, the a n h y d r o - l i n k a g e is cleaved in T20/TCI. The formation of the e t h e r - l i n k a g e between C 4 and C 9 is p a r a l l e l e d by an e p i m e r i z a t i o n at C4 (Goto and others, 1965). Since this carbon atom is part of a ring system, the e p i m e r i z a t i o n was expected to e n c o u n t e r c o m p a r a b l y stable hydrogen incorporation. The main a d v a n t a g e of this sequence of known reactions (Tsuda and others, 1964) is the p h y s i o l o g i c a l l y inactive a n h y d r o intermediate. Therefore, chemical c o n d i t i o n s can be o p t i m i z e d in the milligram range using bioassays as an analytical tool. After q u a n t i t a t i v e reaction of initial TTX, an overall yield of ca. I % was o b t a i n e d for recovered TTX. The specific a c t i v i t y of this p r e p a r a t i o n is 18 000 Ci/mol, which is not far from ca. 30 O00 Ci/mol e x p e c t e d for the i n c o r p o r a t i o n of one tritium into one m o l e c u l e of TTX. In the course of months, no back exchange has been obse r v e d in the range of p h y s i o l o g i c a l conditions. Binding
of 3 H - T e t r o d o t o x i n
to N a - C h a n n e l s
For binding studies in vitro, m e m b r a n e fragments from the electric organ of E l e c t r o p h o r u s electricus were p r e p a r e d by d i f f e r e n t i a l and isopycnic d e n s i t y gradient c e n t r i f u g a t i o n (Kasai and Changeux, 1971a). Specific binding being d i s p l a c e d by 10 -6 M reference TTX shows h a l f - s a t u r a t i o n at a c o n c e n t r a t i o n of (20 + 10) nM 3H-TTX. Bioassay with frog nodes reveals an apparent d i s s o c i a t i o n c o n s t a n t of 3 nM in vivo (Gr~nhagen and colleagues, 1980). To prove the higher apparent d i s s o c i a t i o n constant for E l e c t r o p h o r u s e l e c t r i c u s m embr a n e fragments, the c o m p e t i t i o n b e t w e e n 3H-TTX and reference TTX was analyzed. D i s p l a c e m e n t of 3H-TTX by reference TTX is in q u a n t i t a t i v e agreement with the apparent d i s s o c i a t i o n constant of KD = (20 + 10) nM. F u r t h e r more, these e x p e r i m e n t s c o n f i r m e d the c o n c e n t r a t i o n of 3HZTTX as d e t e r m i n e d by bioassay. Thus the apparent d i s s o c i a t i o n c o n s t a n t K D = 3 nM for b l o c k i n g N a - c u r r e n t s in frog nodes in vivo is d e f i n i t e l y much lower than the a p p a r e n t d i s s o c i a t i o n c o n s t a n t for h a l f - s a t u r a t i o n of binding to N a - c h a n n e l s in E l e c t r o p h o r u s electricus m e m b r a n e fragments. This d i f f e r e n c e may be due in part to a high density of negative surface charges in nodal regions (Drouin
Na-Channels in Membrane Fragments
and Neumcke, 1974), w h i c h charged TTX at the node.
increases
the local c o n c e n t r a t i o n
75
of p o s i t i v e l y
To o b t a i n more insight into the m o l e c u l a r basis of TTX binding, the k i n e t i c s of T T X - a s s o c i a t i o n and d i s s o c i a t i o n were measured. Both p r o c e s s e s were o b s e r v e d at the r e s o l u t i o n limit of M i l l i p o r e f i l t r a t i o n techniques. At 0 oc the b i m o l e c u l a r forward rate c o n s t a n t was kf ~ 3 x 105 M - I s e c -I as d e t e r m i n e d from the initial slope (GrHnhagen, Dahl and Reiter, 1980). D i s s o c i a t i o n always showed a fast process with k~ = 2.5 x lO-2sec -1, in some p r e p a r a t i o n s a m i n o r part of T T X b i n d i n g d i s s o c i a t e d more slowly with k6 ~ IO-3sec -I (Fig. I). U s i n g the kinetic c o n s t a n t s o b s e r v e d for the m a j o r part of TTX binding, a t h e r m o d y n a m i c d i s s o c i a t i o n c o n s t a n t of K D ~ 8 x 10 -8 M is calculated, w h i c h is not in c o n t r a d i c t i o n to the value d e r i v e d from c o n c e n t r a t i o n d e p e n d e n t s a t u r a t i o n of binding. I n t e r e s t i n g l y the forward b i n d i n g c o n s t a n t 1.5
TTX • 2OhM kl b = 0,025sec-1(80%)
3000-
kl~ = 8.3 x I0 "/' sec -I (20%} E • LO
2000
erl
o
\
"~"
m
% -0.5
I.ooo
°
~
o
Time [mini
Fig.
I. D i s s o c i a t i o n k i n e t i c s of 3H-TTX. Difference between total b i n d i n g and u n d i s p l a c e d b i n d i n g in the p r e s e n c e of I ~M r e f e r e n c e TTX. M e m b r a n e fragments (i.iO g/ml sucrose density) were i n c u b a t e d w i t h 20 nM 3H-TTX. The full line is c a l c u l a t e d as a s u p e r p o s i t i o n of two d i s s o c i a t i o n p r o c e s s e s with k~ = 2.5 x iO-2sec -I (80 % of the initial amplitude) and k6 = 8.3 x 10 -4 sec -I (20 % of the initial amplitude).
kf is c o n s i d e r a b l y b e l o w a d i f f u s i o n c o n t r o l l e d value. F o r w a r d a s s o c i a t i o n slow c o m p a r e d to in vivo data results also for o t h e r b i o c h e m i c a l preparations, where a p p a r e n t t h e r m o d y n a m i c d i s s o c i a t i o n c o n s t a n t s in the n a n o m o l a r range and k i n e t i c d i s s o c i a t i o n p r o c e s s e s in the m i n u t e time range were found (Krueger and others, 1979, Tab. 3). Identical t h e r m o d y n a m i c d i s s o c i a t i o n c o n s t a n t s in vivo and in vitro have been taken as e v i d e n c e for p r e s e r v e d m o l e c u l a r p r o p e r t i e s of the T T X - r e c e p t o r . If i d e n t i t y results from c o m p e n sation of slowed down forward- and backward-kinetics, this a r g u m e n t is not convincing. A n a l y s i s of the b i n d i n g k i n e t i c s may t h e r e f o r e reveal more insight into this part of the N a - c h a n n e l .
76
H.H. Griinhagen
P u r i f i c a t i o n of N a - C h a n n e l Rich Membrane F r a ~ m e n t s If the electric organ of E l e c t r o p h o r u s e l e c t r i c u s is h o m o g e n i z e d by a VirTis, and the low speed supernatant f r a c t i o n a t e d on a sucrose d e n s i t y - g r a d i e n t , the plasma m e m b r a n e markers are e n r i c h e d at d e n s i t i e s between ca. 1.04 and 1.16 g/ml (Kasai and Changeux, 1971a, 1971b, 1971c; Reed and Raftery, 1976; Baumann, C h a n g e u x and Benda, 1970). C o n c e r n i n g s p e c i a l i z a t i o n s of the plasma m e m b r a n e (innervated versus non innervated face, subsynaptic versus extrasynaptic areas, planar regions versus invaginations) there is, however, a c o m p a r a b l y broad d i s t r i b u t i o n of m a r k e r s w i t h i n this range of sucrose densities and an u n a m b i g u o u s c o r r e l a t i o n b e t w e e n d e n s i t y fractions in vitro, and m e m b r a n e sections in vivo is not obvious (Rosenberg and others, 1977). In vivo, N a - c h a n n e l s are likely to be located in e x t r a s y n a p t i c regions of the innervated face. In striated muscle, N a - c h a n n e l s have been functionally detected in transverse tubules (Costatin, 1975), which may well correspond to deep transverse i n v a g i n a t i o n s on the innervated face of the e l e c t r o p l a q u e (Bourgeois and others, 1978). Therefore, our p r e p a r a t i v e efforts aim at a p u r i f i c a t i o n of e x t r a s y n a p t i c m e m b r a n e fragments bearing c h a r a c t e r i s t i c s of the planar or of the i n v a g i n a t e d regions of the innervated face.
TABLE
I
Specific Binding in M e m b r a n e F r a c t i o n s Specific Binding [ p m o l / m ~ 3H-TTX
Density gradient fraction (1.O6 g/ml)
2.6
e-neurotoxin 1.0
after electrophoresis: Zone Zone Zone zone
I II III IV
(cathode)
(anode)
(I 41 28 (I
6.1 22 24 3
Specific binding for s a t u r a t i n g c o n c e n t r a t i o n s of neurotoxins. 3 H - T T X - b i n d i n g is c o r r e c t e d for loss during filtration. Specific binding after electrophoresis represents peak values in I ml fractions.
Using shallow sucrose gradients (Gr~nhagen, Dahl and Reiter, 1980) electrophoretic separations of density g r a d i e n t fractions were obtained with a c a r r i e r - f r e e column technique. T y p i c a l results are shown in Table I. Density gradient fractions in most p r e p a r a t i o n s have specific b i n d i n g of 3H-TTX and of e - n e u r o t o x i n ranging from I to 5 pmol/mg. In the density range of interest (1.O4 to 1.16 g/ml) , e l e c t r o p h o r e s i s yields a f r a c t i o n a t i o n into 4 distinguishable zones. The zone with the highest specific binding of 3H-TTX, i.e. zone III, reveals i n t e r e s t i n g l y a c o m p a r a b l y high specific binding of ~neurotoxin. Qualitatively, c o e x i s t e n c e of the receptors for both types of n e u r o t o x i n s is expected for e x t r a s y n a p t i c regions. Quantitatively, 28 p m o l / m g c o r r e s p o n d to ca. 75 T T X - r e c e p t o r s per square micrometer, if as a first a p p r o x i m a t i o n a m e m b r a n e thickness of 75 ~, a p r o t e i n - t o - l i p i d ratio of 1.5 and a density of I g/ml are assumed. A s s u m i n g furthermore a m o l e c u l a r w e i g h t of 300 OOO for the N a - c h a n n e l and a 1:1 s t o i c h i o m e t r y between T T X - r e c e p t o r and Na-channel, the channel w o u l d r e p r e s e n t 0.8 % of the total protein in these p u r i f i e d m e m b r a n e fragments. In vivo, m o r p h o l o g i c a l analyses have allowed to d e t e r m i n e the density of e - n e u r o t o x i n - r e c e p t o r s in e x t r a s y n a p t i c regions of the innervated face (Bourgeois and others, 1978). A value of 370 ~ 260 um -2 was found for this part of the plasma m e m b r a n e including the invaginations. Using the data for specific binding from Tab. I to estimate the receptor densities, one may conclude, that e l e c t r o p h o r e s i s - z o n e III i) q u a l i t a t i v e l y reveals the c h a r a c t e r i s t i c s of excitable e x t r a s y n a p t i c
Na-Channels in Membrane Fragments
77
m e m b r a n e fragments and appears to r e p r e s e n t ii) q u a n t i t a t i v e l y a p r e p a r a t i o n c o n t a i n i n g 15 to 50 % pure e x t r a s y n a p t i c e x c i t a b l e m e m b r a n e fragments, V e s i c u l a r M o r p h o l o g y of M e m b r a n e F r a g m e n t s and C o n d i t i o n s Ion-Flux
for Specific Na-
Freeze fracture e l e c t r o n m i c r o g r a p h s (prepared by G. Dahl) d e m o n s t r a t e that the m a j o r part of m e m b r a n e fragments in d e n s i t y g r a d i e n t fractions forms v e s i c l e s w i t h d i a m e t e r s from i000 to 5000 ~. W h e n the cells are homogenized, their outer surface may a priori become the outer or the inner surface of a vesicle. Since m e m b r a n e area in i n v a g i n a t i o n s r e p r e s e n t s ca. 90 % of the total e x t r a s y n a p t i c area (Bourgeois and others, 1978) and since these tubules may tend to preserve their inverse o r i e n t a t i o n w h e n homogenized, a c o n s i d e r a b l e p r o p o r t i o n of v e s i c l e s could have an i n s i d e - o u t orientation. To a n a l y z e the o r i e n t a t i o n of v e s i c l e membranes, their b i n d i n g of extrac e l l u l a r l y e x p o s e d g l u c o s i d e - or m a n n o s i d e - m o i e t i e s to s e p h a r o s e - b o u n d conc a n a v a l i n A columns was studied (Edelman, Rutishauser, and Milette, 1971). A f r a c t i o n b r e a k i n g through the column w i t h Ringer e l u t i o n has lower specific b i n d i n g for outer surface m a r k e r s (i.e. 2.6 p m o l / m g for TTX) than the d e n s i t y g r a d i e n t f r a c t i o n a p p l i e d to the column (i.e. 3.6 pmol/mg). P r e s u m a b l y this f r a c t i o n has an e n r i c h e d content of i n s i d e - o u t vesicles. The g l u c o s i d e eluted fraction has highly e n r i c h e d specific b i n d i n g of the outer surface markers TTX (up to 26 pmol/mg) and ~ - n e u r o t o x i n (up to 50 pmol/mg). However, with 6 % it r e p r e s e n t s only a m i n o r p r o p o r t i o n of the r e c o v e r e d material. D e p e n d e n t on the a t t r i b u t i o n of i r r e v e r s i b l y c o l u m n - b o u n d m a t e r i a l (ca. 50 %) to the i n s i d e - o u t or o u t s i d e - o u t population, the i n s i d e - o u t vesicle fraction may be e s t i m a t e d to r e p r e s e n t 40 to 90 % of d e n s i t y g r a d i e n t material. These high values are in a g r e e m e n t w i t h the a b u n d a n c e of t r a n s v e r s e tubules on the i n n e r v a t e d face. A c c o r d i n g l y , i n s i d e - o u t v e s i c l e s could be an e n r i c h e d pop u l a t i o n of m e m b r a n e fragments from t r a n s v e r s e tubules. V e s i c l e s from the e l e c t r i c organ of E l e c t r o p h o r u s e l e c t r i c u s may be used for ion flux studies in vitro. I n v e s t i g a t i o n of specific ion flux through synaptic c h a n n e l s has r e s u l t e d in important c o n t r i b u t i o n s to an unders t a n d i n g of the a c e t y l c h o l i n e - r e c e p t o r (Kasai and Changeux, 1971; Hess, Cash and Aoshima, 1979; Cash and Hess, 1980; Aoshima, Cash and Hess, 1980). In a c o m p a r a b l e way it has been p o s s i b l e to observe N a - f l u x t h r o u g h N a - c h a n n e l s in m i x e d v e s i c l e s from lobster nerve and soybean liposomes, where c h a n n e l s were o p e n e d w i t h v e r a t r i d i n e and b l o c k e d s p e c i f i c l y with t e t r o d o t o x i n (Villegas and coworkers, 1977, 1979). In m e m b r a n e v e s i c l e s from E l e c t r o p h o r u s e l e c t r i c u s e l e c t r i c organ we have no e v i d e n c e for specific effects of N a - c h a n n e l n e u r o t o x i n s on Na-efflux, if v e s i c l e s are e q u i l i b r a t e d inside and outside w i t h Ringer s o l u t i o n (160 mM NaCI, 5 m M KCI, 2 m M CaCI2, 2 m M MgCI2, 1.5 m M p h o s p h a t e buffer, pH 7.0; "Na-Ringer"), a g r a d i e n t e x i s t i n g only for tracer amounts of 22Na. Specific n e u r o t o x i n s were, however, found to control 2 2 N a - e f f l u x from Electrophorus e l e c t r i c u s vesicles, if the v e s i c l e s were e q u i l i b r a t e d before the flux e x p e r i m e n t in N a - R i n g e r and t r a n s f e r r e d at the b e g i n n i n g of the efflux into K - R i n g e r (160 m M KCI, 5 mM NaCI, 2 m M CaCI2, 2 m M MgCI2, 1.5 m M p h o s p h a t e b u f f e r pH 7.0), or vice versa. In such experiments, the o p e n i n g of the Nachannel was p r o l o n g e d by c e v a d i n e (HonerjMger, 1973) or by t r a n s i e n t treatment of i n s i d e - o u t v e s i c l e s w i t h N - b r o m o - a c e t a m i d e (NBA) (Oxford, Wu and Narahashi, 1978). Fig. 2 shows an efflux e x p e r i m e n t after t r e a t m e n t of the v e s i c l e s w i t h NBA. In the p r e s e n c e of TTX, the e f f l u x curve is s i g n i f i c a n t l y shifted to higher amounts of 22Na r e t a i n e d in the v e s i c l e s in this time range. The time r e s o l u t i o n of f i l t r a t i o n t e c h n i q u e s does not a l l o w to m o n i t o r the initial phase of efflux, where the slope of the e f f l u x curve is e x p e c t e d to be steeper in the absence of TTX. An e s t i m a t i o n shows that an e s s e n t i a l part of 22Naefflux may well occur in the m i l l i s e c o n d time range: under conditions as
78
H.H.
Gr{inhagen
NBA, Imin, ImM
OW
MF + TTX o MF x Filters
•
3
o 04 04
"O O
.c_ o O
n.
X~X-...
0
x
i
I
i
I
30
60
90
120
Time [sec] Fig.
2. 2 2 N a - e f f l u x from vesicles (l.ll g/ml sucrose density) t r a n s i e n t l y treated with N - b r o m o a c e t a m i d e (NBA, I min at a c o n c e n t r a t i o n of I mM). Incubation o v e r n i g h t in Na-Ringer, vesicles trapped on a c o m b i n a t i o n of G F / D - p r e f i l t e r and a HAWP M i l l i p o r e - f i l t e r (13 mm), c o n t r o l l e d elution of r el e a s e d tracer with K - R i n g e r (500 ~i per elution step). Mean values from 3 experiments.
chosen for the e x p e r i m e n t in Fig. 2, 160 mM NaCI bulk c o n c e n t r a t i o n and ca. 3 % i n t r a v e s i c u l a r volume y i e l d ca. 5 m M i n t r a v e s i c u l a r Na-ions. A specific T T X - r e c e p t o r c o n c e n t r a t i o n of 2.5 pmol/mg protein and a p r o t e i n c o n c e n t r a t i o n of 2 mg/ml during the e q u i l i b r a t i o n period c o r r e s p o n d to ca. 5 nM Na-channels, if a 1:1 s t o i c h i o m e t r y is assumed for T T X - r e c e p t o r and Na-channels. C o m p a r i n g 5 mM i n t r a v e s i c u l a r N a - i o n s w i t h 5 nM N a - c h a ~ n e l s , a number of 106 Na-ions per channel is obtained. This compares to iO ions passing a single Na-channel per second. The o c c u r r e n c e of specific n e u r o t o x i n effects appears to require d i f f e r i n g Na- and K-ion c o n c e n t r a t i o n s inside and outside the vesicles. One may h y p o t h e s i z e that a m e m b r a n e p o t e n t i a l has to be e s t a b l i s h e d before Nachannels can be e f f i c i e n t l y opened. This c o n d i t i o n may be related to HodgkinHuxley i n a c t i v a t i o n (Hodgkin and Huxley, 1952) or to slower m e c h a n i s m of ina c t i v a t i o n (Brismar, 1977; Neumcke, Schwarz and St~mpfli, 1979). A l t e r n a t i v e ly, asymmetric b i n d i n g of Na- and K-ions to specific sites on o p p o s i t e faces of the m e m b r a n e c o u l d also a c c o u n t for the o b s e r v e d phenomena. The o c c u r r e n c e ofa n e u r o t o x i n effect i n d e p e n d e n t on the d i r e c t i o n of Na- and K - g r a d i e n t s is
Na-Channels in Membrane Fragments
79
probably due to the heterogeneity of the preparation, which contains both outside-out and inside-out vesicles.
CONCLUSIONS Our method to tritiate TTX by a defined chemical procedure yields a preparation of 3H-TTX with a high specific activity. This specific ligand allows us to identify Na-channel-rich membrane fragments in vitro and to study binding kinetics. In density gradient fractions, the specific binding of 3H-TTX is comparable to the specific binding of a-neurotoxins. Correspondingly, this membrane preparation may be as useful for biochemical studies of Na-channels as it has turned out to be for investigations of the acetylcholine-receptor (Kasai and Changeux, 1971a, 1971b, 1971c; Hess, Cash and Aoshima, 1979). Further purification of membrane fragments is possible by carrier-free column electrophoresis. This technique yields fractions with specific binding of TTX and ~-neurotoxin close to values expected for highly purified extrasynaptic fragments of the excitable face of the electroplaque (Bourgeois and colleagues, 1978). Such a preparation is of interest for spectroscopic studies, where high densities of receptors are of major importance. The vesicular morphology of most membrane fragments is of high interest for functional studies in vitro. A high proportion of vesicles in density gradient fractions is inside-out. Although this heterogeneity may complicate a quantitative analysis of ion flux data, after purification outside-out and insideout fractions of vesicles will certainly be useful for studies of vectorial phenomena and sidedness. Specific effects of neurotoxins on ion flux were observed, when Na- and Kion concentrations were modified between equilibration with the tracer and start of an efflux experiment. Further experiments have to analyze the molecular basis of this phenomenon to allow us to correlate molecular and functional data.
ACKNOWLEDGEMENT Part of the data have been obtained in cooperation with Drs. H. Fasold, G. Dahl, M. Rack and R. St~mpfli. The support of this work by V. Ullrich and R. St~mpfli and the expert assistance of W. BHhler, P. Gries, P. Reiter and R. Stolz are gratefully acknowledged. Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 38 "Membranforschung".
REFERENCES Abita, J.P., R. Chicheportiche, H. Schweitz, and M. Lazdunski (1977). Biochemistry, 16, 1838-1844. Agnew, W.S., S. R. Levinson, J. S. Brabson, and M. A. Raftery (1978). Proc.Natl.Acad. Sci.USA, 75, 2606-2610. Agnew, W. S., A. C. Moore, S. R. Levinson, and M. A. Raftery (1980). Biochem. Biophys. Res.Comm., 92, 860-866. Aoshima, H., D. J. Cash, and G. P. Hess (1980). Biochem. Biophys.Res.Comm., 9_22, 896-904. Armstrong, C. M. (1975). Quart. Rev.Biophys.,[, 179-210. Balerna, M., M. Fosset, R. Chicheportiche, G. Romey, and M. Lazdunski (1975). Biochemistry , i_44, 55OO-5511. Barnola, F. V., R. Villegas, and G. Camejo (1973). Biochim. Biophys.Acta, 298, 84-94. Barnola, F. V., and R. Villegas (1976). J.Gen. Physiol., 67, 81-90. de Barry, J., M. Fosset, and M. Lazdunski (1977). Biochemistry, 16, 38503855.
80
H.H. Gr~inhagen
Bauman, A., J. P. Changeux, P. Benda (1970). FEBS Lett., 8, 145-148. Bourgeois, J. P., J. L. Popot, A° Ryter, and J. P. C h a n g e u x (1978). J. Cell Biology, 79, 2OO-216. Brismar, T. (1977). J. Physiol. London, 270, 283-297. Catterall, W. A. (1975). Proc.Natl.Acad. Sci.USA, 72, 1782-1786. Cash, D. J., and G. P. Hess (1980). Proc.Natl.Acad. Sci.USA, 77, 842-846. Changeux, J.P., M. Kasai, and C. Y. Lee (1970). Proc.Natl.Acad. Sci.USA, 67, 1241-1247. Costatin, L. L. (1975). F e d e r a t i o n Proc., 34, 1390-1394. Couraud, F., H. Rochat, and S. Lissitzky (1980). Biochemistry, 19, 457-462. Drouin, H., and B. Neumcke (1974). Pfl~gers Arch., Eur.J.Physiol., 351, 207-229. Edelman, G. M., U. Rutishauser, and C. F. Milette (1971). Proc.Natl.Acad. Sci.USA, 68, 2153-2157. Goto, T., Y. Kishi, S. Takahashi, and Y. Hirata (1965). Tetrahedron, 21, 2059-2088. Gr~nhagen, H. H., M. Rack, R. St~hapfli, H. Fasold, and P. Reiter (submitted for publication). Gr~nhagen, H. H., G. Dahl, and P. Reiter (1980). In preparation. Gr~nhagen, H. H. (1980). In preparation. Hess, G. P., D. J. Cash, and H. A o s h i m a (1979). Nature, 282, 329-331. Hodgkin, A. L., and A. F. Huxley (1952). J. Physiol. (London), 117, 500-544. Honerj~ger, P. (1973). N a u n y n - S c h m i e d e b e r g ' s Arch. Pharmacol.,280, 391-416. Huxley, A. F., and R. St~mpfli (1949). J. Physiol. (London), 108, 315-339. Kasai, M., and J. P. C h a n g e u x (1971). J. Membr. Biol., 6, 1-23. Kasai, M., and J. P. C h a n g e u x (1971). J. Membr. Biol., 6, 24-57. Kasai, M., and J. P. C h a n g e u x (1971). J. Membr. Biol., ~, 58-80. Keynes, R. D. (1975). In The Nervous System, D. B. Tower, Ed., Vol. I: The Basic Neurosciences, pp. 165-175, Raven Press, New York. Keynes, R. D. (1979). Sci. Amer., 240, 98-107. Krueger, B. K., R. W. Ratzlaff, G. R. Strichartz, and M. P. Blaustein (1979). J. Membr. Biol.,50, 287-310. Miledi, R., P. Molinoff, and L. Potter (1971). Nature, 229, 554-557. Nachmansohn, D. (1963). In Handbuch der e x p e r i m e n t e l l e n Pharmakolo@ie, Erg ~ n z u n g s w e r k XV (0. Eichler, A. Farah, Eds.) pp. 701-740, Springer, Berlin. Narahashi, T. (1974). Phys. Rev., 54, 813-889. Neumcke, B., W. Schwarz, and R. St~mpfli (1979). Biochim. Biophys.Acta, 558, 113-118. Oxford, G. S., C. H. Wu, and T. N a r a h a s h i (1978). J. Gen. Physiol., 71, 227-247. Reed, J. K., and M. A. Raftery (1976). Biochemistry, 15, 944-953. Ritchie, J. M., R. B. Rogart, and G. Strichartz (1976). J. Physiol. (London), 261, 477-494. Ritchie, J. M., and R. B. Rogart (1977). Rev.Physiol. Biochem. Pharmacol., 79, 1-50. Rosenberg, P., I. Silman, E. Ben-David, A. DeVries, and E. Condrea (1977). J. Neurochemistry, 29, 561-578. Tsuda, K., S. Ikuma, M. Kawamura, R. Tachikawa, K. Sakai, C. Tamura, and O. A m a k a s u (1964). Chem. Pharm. Bull., i_22, 1357-1374. Villegas, R., G. M. Villegas, F. V. Barnola, and E. Racker (1977). Biochem. Biophys. Res. Comm., 79, 210-217. Villegas, R., G. M. Villegas, F. V. Barnola, and E. Racker (1979). In Advances in C y t o p h a r m a c o l o ~ y (B. Ceccarelli, F. Clementi, Eds.) Vol. 3, pp. 373-385, Raven Press, New York.