Monoclonal antibodies associated with sodium channel block nerve impulse and stain nodes of Ranvier

168

Brain Research, 310 (1984) 168-173 Elsevier

BRE 20368

Monoclonal antibodies associated with sodium channel block nerve impulse and stain nodes of Ranvier HAMUTAL MEIRP*, IRENE ZEITOUN 1, HANS H. GRUNHAGEN 2, VARDA LEV-RAM 3, ZELIG ESHHAR 3 and JOSEPH SCHLESSINGER 3 ~Department of Physiology and Pharmacology, The Sackler School of Medicine, Tel-Aviv University, Ramat A viv 69978 (Israel), 21nstitute of Physiological Chemistry, UniversitdtHomburg, D-6650Homburg/Saar (F. R. G.) and 3The Weizmann Institute of Science, Rehovot 76100 (Israel)

(Accepted May 8th, 1984) Key words: monoclonal antibodies - - sodium channels - - node of Ranvier - - conduction block - - electroplax - - neurotoxins

Monoclonal antibodies were generated against native eel electroplax sodium channels in their natural membrane. These antibodies block nerve conduction in rat central (optic) and peripheral (sciatic) nerve. The antibody binding to eel electroplax membrane fragments and to rat brain synaptosomes can be modulated by neurotoxins. Thus it implies that active sites of the sodium channels are immunogenic in their natural membrane. Unlike the antibodies described in the past, our antibodies recognize antigenic determinants which are associated with the physiological activity of the channel and have been conserved through evolution.

The majority of the mature neurons are capable of generating and conducting electrical impulses 16. This ability depends upon the presence and activity of specific ionic channels and permeabilities 6,16:8.19. The voltage d e p e n d e n t sodium channel plays an important role in the generation of the action potential. This channel is gated by the t r a n s - m e m b r a n e voltage through resting, active and inactive states, thereby generating the early steps in nerve excitation and conduction2:5:6. The nature of the sodium channel has been investigated by various experimental approaches including toxin binding assays0:8:9 and biochemical purification 1,3,9,14. The binding of neurotoxins revealed the allosteric nature of the sodium channels. T e t r o d o t o x in (TTX) and saxitoxin block the flux of sodium ions through the channels, presumably by interfering with the so-called 'selectivity filter' site 15,22. The alkaloids, lipid-soluble toxins such as veratridine, batrachotoxin and others cause a persistent activation of sodium flux through the channel and prolong membrane depolarization, presumably by altering the sodium channel ionophore 6.1s. The p o l y p e p t i d e toxins

from the venom of scorpion (Leirus, Androctonus) and of a n e m o n e most likely interact with the 'inactivation gating site '6:s, thus increasing the duration of the action potential which in turn potentiates sodium flux through the o p e n e d channel. Finally, polypeptide toxins derived from Tityus and Centroides were shown to interact with the 'voltage activation gating sites '4 thus producing repetitive firing and oscillation of m e m b r a n e potential by inducing Na+-current at sub-threshold m e m b r a n e potentials. A versatile a p p r o a c h to study the location, structure-function relationship and the mechanism of channel conductance involves the application of specific antibodies directed against the channel 25. This approach has previously been utilized10.11, z0 using polyclonallO, tl and recently m o n o c l o n a p l , 20 antibodies generated against the solubilized sodium channell,9 p r e p a r e d from the eel electroplax m e m b r a n e . These antibodies recognize the solubilized channel isolated from the eel electroplaxl0:l, 20 and also stain the channel in the m e m b r a n e of eel peripheral nerve 10,11. Hence, these antibodies are species specific, recognizing only antigenic determinants of the fish sodium

* Correspondence: H. Meiri, Department of Physiology and Pharmacology, The Sackler School of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel.

0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

169 channel. The immunogen used in the present study was a membrane preparation from the eel electroplax, Which is a relatively rich source of sodium channel 13. This membrane preparation retains the functional properties of the channel moiety and the capacity to bind specifically various neurotoxins. Therefore, it was anticipated that the antibodies generated against this membrane will recognize specific antigens on the sodium channel which are relevant to its physiological activity. The two monoclonal antibodies described in this study interact with the eel electroplax sodium channel in its membrane-bound form. In addition, they block impulse conduction in the mammalian peripheral (sciatic) and central (optic) nerve, as well as staining the nodes of Ranvier in mammalian peripheral nerve. Furthermore, their binding to the node of Ranvier and to membrane fragments of eel electroplax (EMF) and to membrane vesicles of rat brain synaptosomes (RMV) is modulated by neurotoxins. Our results suggest that these antibodies are directed against the voltage-dependent sodium channel or closely associated sites in mammalian nerve and eel electroplax. The eel electroplax membranes used for immunization contained 4.3 pmol TTX binding sites per mg protein received from the 1.12 g/ml sucrose gradient as previously described by Grunhagen et al. 20. It was diluted 1:10 in PBS (pH 7.2) and 0.13 ml were injected twice (two weeks apart), together with complete Freund's Adjuvant into footpads of CD2F ~ mice. A month later, the mice received an intraperitoneal booster injection of 0.4 ml and were sacrificed 3 days later for fusion experiments. For hybridization, the spleen cells from the immunized mice were fused with NSO/1 (the non-producing myeloma line derived from NS1/1 Ag 4.1) at 5:1 ratio using 51% (v/v) polyethylene glycol 1500 as fusing agent following the procedure of Galfre and Milstein 12. Hybrid cells growing in hypoxanthine-aminopterine-thymidine-selective medium were selected for secreting relevant antibodies. Since our antigen was a relatively crude membrane preparation containing a number of membranebound molecules 13, a specific selection procedure was employed to detect relevant antibodies. Our screening procedure was based upon recognizing the channels according to their ability to bind neurotox-

ins, their location in myelinated axons and their function in conducting electrical impulses. A solid-phase radioimmunoassay (SP-RIA) was used to detect relevant antibodies which bind to the original antigen. Eighty out of 520 hybridomas showed at least fourfold higher binding than the background binding as determined by growth medium of hybridoma and by irrelevant antibodies. In the second assay, we determined the effects of various neurotoxins (TTX, veratridine, and scorpion toxin of Leirus) on the binding of the antibodies to the membrane. Among 80 hybridomas tested only 12 were scored as ' E M F positive' since their binding was either significantly decreased or increased in the presence of at least one of the neurotoxins. Then, membrane vesicles of rat brain synaptosomes (RMV 17) - - containing a similar number of TTX binding sites as EMF - - were used as antigen for the SP-RIA. In this experiment only 7 hybridomas, out of the 80 'positive' clones (see above), secreted antibodies whose binding was modulated by neurotoxins. These 7 clones were also included in the 12 'EMF positive' clones. Moreover, neurotoxins modulated their binding to EMF and RMV in a similar manner and potency when tested by a similar antibodies concentration. The 80 'positive' hybridomas were checked for their ability to stain nodes of Ranvier in rat sciatic nerve. Only 5 hybridomas were scored 'node positive', all of them are also ' R M V positive'. These 5 positive hybrid cultures were cloned in soft agar. Hybridomas from isolated clones were grown in culture and ascites produced in CD2F 1 mice. For all assays described below monoclonal Ig were isolated from media of cultured hybridoma or from ascites fluid by a 40% ammonium sulfate precipitation. One monocional antibody (mAb) denoted SC-66-5 (IgG2) bound to both EMF and RMV. The binding of the antibodies was reduced by an excess of either veratridine (100 ~ M - 1 mM) or scorpion toxin of Leirus 5 (1-10/~M), but was not affected by TTX (100 /~M-1 mM) as summarized in Table I. The partial inhibition of the m A b binding to both EMF and RMV by veratridine or by Leirus toxin could be due to the fact that only a part of the sites occupied by the antibodies is associated with the binding of these toxins. These toxins bind to two different sites 6,8A9, both are associated with the channel-gating activity. Thus, it is expected that these mAbs may

170 TABLE I Modulation of antibodies binding by neurotoxins

The binding of antibodies to the sodium channel-enriched membrane fragments from rat brain synaptosomes (RMV) and eel electroplax membrane fragments (EMF) in the solid-phase radioimmunoassay (SP-RIA). Disposable microtiter plates (flexible PVC; Cook product) were coated with poly-L-lysin(PLL), (10/~g/ml saline) for 30 min at room temperature. Excess of PLL was removed and EMF or RMV containing 500-1000 fmol TTX binding sites in PBS, pH 7.4, were added to each well for 1 h at room temperature followed by 24 h at 4 °C in a moisture container. The wells were washed with excess of PBS containing 0.01% NaN3 and 1 mg/ml BSA (PBS-BSA), and they were then re-filled with excess of PBS-BSA for 30 min at room temperature to block the remaining sites on the wells. They were then incubated with 10/A of PBS (control) or of 50 #M TTX (Cal-Biochem) or 500/~M veratridine (Sigma) or 5 ~M scorpion toxin (Sigma) for 1 h at room temperature. Then 40/A of hybridoma's supernatant containing antibodies were added for 2-3 h at room temperature. The wells were washed in PBS-BSA 3 more times. Then, a second antibody ([1251]goat anti-mouse Ig, 150,000 cpm in 50/A) was added for 2-3 h at room temperature. The wells were well washed 3 more times and the radioactive content was determined with a gamma counter. Values are the means of triplicates of 3 different experiments. Background radioactivity was determined using supernatant of irrelevant hybridoma belonging to the same fusion experiment (EMF negative, see text) and was 1000 cpm (similar to the value received from hybridoma growth medium - - DMEM-horse serum). Binding values of positive clones ranged between 4000-20,000 cpm. 100% binding for each antibody was the value received in PBS in the absence of neurotoxins after background subtraction. For each combination of antibodies-antigen, the percent binding was the ratio between values obtained in the presence of neurotoxins versus the value obtained in their absence. SC-17 represents a control of 'positive' hybridoma (see text) derived from the 80 hybridoma positive (see text) which bind to both EMF and RMV, but its binding is not affected by any of these toxins. The isotype of the antibodies was determined by immuno-diffusion with mono-specificantisera (Meloy). Monoclonal antibodies no.

SC-66-5 SC-72-14 SC-17

Antibody class

IgG 1 IgG2

Percent binding to the antigen in presence of: TTX (IO pM) 'selectivity filter'

Veratridine (lOOpM) 'activation gating'

Scorpion toxin (1 pM) 'inactivation gating'

RMV

EMF

RMV

EMF

RMV

EMF

89+ 11 99 _+ 13 95_+11

80+ 12 85 _+ 11 107_+13

51 _+7 70 _+ 10 96_+7

66_+ 15 58 _+ 13 117_+11

48_+ 14 202 -+ 21 90_+13

42_+ 11 158 +_ 13 95_+10

somehow interfere with this activity. Neurotoxins inhibition of antibodies binding argues against a possible association of these antibodies with other neighboring m e m b r a n e molecules, such as Na+/K +ATPase8, 26,27 or potassium channels that do not bind these toxins. A n o t h e r line of evidence to identify the antigenic site of these antibodies emerges from electrophysiological experiments. These antibodies block completely and in a similar m a n n e r the c o m p o u n d action potential of rat sciatic nerve and optic nerve (Fig. 2) although the last is more sensitive. Within 10-20 min following bath application of 2-4/~g m A b / m l on optic nerve (Fig. 2 A - C ) or 50-100/~g m A b / m l on sciatic nerve, the antibodies attenuated the early phase of depolarization associated with fast conducting fibers. This was followed (in 10-20 min) by a prolongation of the repolarization phase associated with slow conducting axons. Since two toxins, working on two different sites of the sodium channel, inhibit the binding of the antibodies, it raises the possibility that the antibodies' binding sites are somehow associated with two aspects of the channel activity. For instance, the

antibodies may decrease channel conduction, thereby leading to the attenuation in the amplitude of the action potential along with increasing channel opening time, thereby prolonging the duration of the attenuated impulse. A voltage clamp analysis is now in progress to distinguish between these and other possible mechanisms. The effect of the antibodies is partially reversible when washout is started immediately after reaching a complete conduction block, implying that conduction block is not the result of a simple deterioration of the neuronal m e m b r a n e . W h e n washout is delayed by 1 - 2 h or when higher m A b dose is applied, the nerve blockage is usually irreversible. A n o t h e r clone, which belongs to the same fusion experiment (SC-4-10, Fig. 2D) binds to the m e m b r a n e , but its binding is not associated with a conduction block. Finally, an indirect immunofluorescent technique was employed to test the ability of clone SC-66-5 to stain the channel in the rat sciatic nerve (Fig. 1). It has previously been reported that the nodal membrane domain is highly enriched by sodium channels 21,28. The immunofluorescence pattern visualized

171

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the specific staining was a b o l i s h e d by v e r a t r i d i n e (Fig. 1B, B ' ) and s c o r p i o n toxin (not s h o w n ) showing that b o t h staining and b i n d i n g are m o d u l a t e d by n e u r o t o x i n s in a similar m a n n e r . In addition, the specific staining of the n o d e of R a n v i e r was o b s e r v e d s u b s e q u e n t to a c u t e d e m y e l i n a t i o n by lysolecitin and o s m o t i c s h o c k t r e a t m e n t 7. C o m b i n i n g t h e results of binding e x p e r i m e n t s , electrophysiological r e c o r d i n g and i m m u n o f l u o r e s c e n c e staining, o n e m a y c o n c l u d e that t h e s e m A b s are associated with site(s) which are an i n t e g r a l part of the sod i u m c h a n n e l o r at least closely a s s o c i a t e d with it. F u r t h e r e v i d e n c e for t h e efficiency of o u r m e t h o d

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Fig. 1. Immunofluorescence staining of nodes of Ranvier with monoclonal antibodies of SC series. Rat sciatic nerve was removed fresh into Locke solution 7 and the perineureum was removed mechanically. Single axons were isolated, connected to thicker bundle on one side and mounted on coverslips. They were incubated for 30 min with goat anti-mouse Ig (20/xg/ml, affinity purified) in PBS (pH 7.4) to block irrelevant sites. Then, they were washed well with PBS, incubated with monoclonal antibodies (100-300/xg/ml) for 1 h, washed well with PBS and then exposed to rhodamine-labeled goat anti-mouse (3.2 mole fluorophore per mole protein, 40/xg/ml) for 20 min. After extensive wash with PBS the nerve was fixed in 3% paraformaldehyde for 30 min, washed well with PBS and mounted for visualization in 90% glycerol in PBS. All steps were performed at room temperature (20-25 °C). A, A': line 66-5. B, B': line 66-5, following preincubation with 100/xM veratridine. C, C': line 66-5, following demyelination with 0.1% lysolecitin (10 min) and osmotic shock (x 5 washes with Locke solution diluted 1:1 with water for 5 min following 5-min wash with Locke solution containing 0.5 M sucrose). D, D': line 72-14. E, E': line 72-14 following preincubation with 100/xM veratridine. Capital letters describe phase micrograph and prime letters are their fluorescent image. No staining was observed with control mAb. with this m A b s h o w e d specific staining of the n o d e s of R a n v i e r . N e i t h e r the i n t e r n o d a l n o r t h e p a r a n o d a l d o m a i n of the axons was s t a i n e d (Fig. 1 A , A ' ) . T h u s the d e t e r m i n a n t s b o u n d by the a n t i b o d i e s are accessible at t h e o u t e r side of the m e m b r a n e . M o r e o v e r ,

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Fig. 2. Blockage of action potential of rat optic nerve. The optic nerve was freshly isolated from mature male rat and incubated for 10 min in Dulbecco's modified Eagle's medium equilibrated with 95% 0 2 + 5% CO 2 in the presence of 0.06% H20 2. It was then transferred to a recording chamber 29 where it was kept in 34-37 °C with constant equilibration of the above-mentioned gas composition. A few drops of fresh medium were added every 5-10 min. Stimulation was provided from a Grass stimulator every second at a strength of 5 V (upper trace in A - C ) or 2.5 V (lower trace in A - C ) through a suction electrode in order to follow fibers with lower or higher threshold. Recording was done with a second suction electrode through a preamplifier to an oscilloscope. When the nerve was blocked, a supra-stimulation was provided (10-20 V) in order to ensure that conduction was completely blocked. A: control. B: 10 min after application of 2.5 ~g of line SC-66-5 added to the recording chamber (2 ml volume). C: the same at 20 min. D: the time course for action potential blockage measured in the presence of clone SC-66-5 and SC-72-14. In addition the results obtained with clone SC-4-10 are demonstrated as values received with a clone which belongs to the 7 'RMV positive' (see text) but does not produce an effect larger than the natural decay which is normally developed in this nerve. Action potential amplitude of different axons was normalized as a percentage of maximum amplitude received in the absence of antibodies.

172 for the generation of monoclonal antibodies against the sodium channel comes from experiments with another antibody obtained from the same fusion experiment, denoted SC-72-14 (IgG1). This mAb binds to EMF and RMV, and its binding is modulated by neurotoxins. Furthermore, a voltage-clamp analysis indicates that it specifically blocks the sodium inward current. I n t h e SP-RIA, the toxins varied in their effects on mAb's binding. While veratridine decreases the binding, Leirus toxin enhances it (Table I). Interestingly, excess of veratridine prevents no more than 50% of mAb's binding. Nevertheless, both toxins, each interacting with a different site of the sodium channel, are associated with the channel-gating mechanism and modulate the binding of SC-72-14. Based on these resuits it is postulated that these mAbs interact with the channel-gating activity. In fact, application of clone SC-72-14 on the optic nerve of rat led to a complete conduction block within 20-60 min (Fig. 2D) depending on the dose (10-100/~g/ml) applied. Again, the optic nerve was affected by lower concentrations than those required for blocking the sciatic nerve (50-200 #g/ml). Clone SC-72-14 attenuated equally both the depolarization and the repolarization phases of the compound action potential. Furthermore, when a voltage-clamp analysis (Meiri et ai. 19a) was performed on a single node of Ranvier of rat sciatic nerve, it was found that these antibodies block the inward sodium current without affecting the outward potassium current or the leakage current. This block is due, primarily, to an increase of the sodium channel inactivation process (a shift in the Hodgkin and Huxley h~ parameter towards hyperpolarization). Thus, voltageclamp analysis reinforced the toxin-modulated SPRIA studies which implied a possible association of these mAbs with the sodium channel gating system. Furthermore, at higher concentrations the antibodies also decreased the maximal channel conductance (gNa) thus providing the basis for mAb's interaction with more than one site at the sodium channel. Eventually, the attenuation of the inward sodium current, as measured by voltage-clamp analysis, led to the blockage of the nerve impulse as measured by extracellular recording. These mAbs also decorate specifically the nodes of Ranvier of rat sciatic nerve (Fig. 1D, D'). This deco-

ration is almost completely abolished following preincubation with veratridine (Fig. 1E, E'). However, unlike the previous clone, clone SC-72-14 also stained the outer surface of the Schwann cell membrane at the paranodal regions. This 'non-specific' staining which is also abolished by veratridine, might be attributed to a number of specific sodium channels, whose existence in Schwann cells has already been demonstrated 23. The strongly stained regions were localized at the node where the density of sodium channel is normally much higher. Unmyelinated axons of sciatic nerve were not stained as it is impossible to visualize their low channel density (approximately 100-200//~m 2) using immunofluorescence microscopy. On the basis of these results, we conclude that the two different antibodies described in this report bind to different sites which are either an integral part of the sodium channel or at least, closely associated with it. Moreover, this study implies that sites associated with the channel activity are immunogenic when the channel is maintained in its membrane. The determinants recognized by the mAbs are accessible at the extracellular side of the membrane and are also preserved throughout the evolution from fish to mammals. Other antibodies, generated by other investigators against the solubilized molecule (the TTX-binding protein associated with the channel) did not induce any physiological activitylO, 11 (also E1lisman and Levinson, personal communication) and were also species specific (or fish specific) 10,11,20. One may conclude that the sodium channel possesses both immunologically conserved and variable regions. The conserved domain appears to be on the external surface of the membrane and could participate in the conduction of the electrical impulses. In addition, there are species-specific domains which are preserved after solubilization of the channel from its native membrane but presumably do not contribute to the channel activity. These antibodies and others are now carefully characterized to gain further insight into the structure-function relationship of the sodium channel molecule. Furthermore, they are excellent probes to detect sodium channel distribution along the neuronal membrane including nerve terminals, dendritic spines, axon hillock, demyelinating axons, biforcating axons and developing axons.

173 W e wish to t h a n k D r . I. R. C o h e n for using his

Smith F a m i l y F o u n d a t i o n (41/81, H . M . ) , t h e Sch-

e l e c t r o p h y s i o l o g i c a l a p p a r a t u s for r e c o r d i n g t h e con-

r i e b e r F o u n d a t i o n for M e d i c a l R e s e a r c h

d u c t i o n of the action p o t e n t i a l in rat o p t i c n e r v e and

N I H G r a n t C A - 2 5 8 2 0 ( J . S . ) and a g r a n t f r o m Stif-

(H.M.),

for his g e n e r o u s a d v i c e a n d e n c o u r a g e m e n t in d e v e l -

tung V o l k s w a g e n w e r k (J.S.). I . Z . is a C h a r l e s E .

oping this m e t h o d o l o g y . T h i s w o r k was s u p p o r t e d by

Smith F a m i l y F o u n d a t i o n f e l l o w s h i p r e c i p i e n t . H . M .

the Israel C e n t e r

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for P s y c h o b i o l o g y ,

Charles

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