Proc. Nat!. Acad. Sci. USAVol. 82, pp. 1842-1846, March 1985Neurobiology

A monoclonal immunotoxin acting on the Na+ channel, withproperties similar to those of a scorpion toxin

(rat brain/electroplax/electrophysiology/binding/Tityus serrulatus)

J. BARHANIN*, H. MEIRIt, G. ROMEY*, D. PAURON*, AND M. LAZDUNSKI**Centre de Biochimie, Centre National de la Recherche Scientifique, Facultd des Sciences, Parc Valrose, 06034 Nice Cedex, France; and tDepartment ofPhysiology and Pharmacology, Tel Aviv University, Sackler School of Medicine, Ramat Aviv 69978, Israel

Communicated by Philip Siekevitz, November 1, 1984

ABSTRACT We describe the properties of a monoclonalantibody against the Na' channel. The antibody, 72.38,competitively inhibited (Ki = 1.5 x 10-9 M) the binding of an'2SI4abeled toxin from the Brazilian scorpion Tityus serrulatus(12'I-TiTXy) to Na' channels of rat brain membranes. Nosignifcant inhibition of binding of a number of other Na'channel toxins was observed. The inhibition of 12'I-TiTXybinding also was observed with the solubilized Na' channelfrom rat brain membranes (K, = 2 x 10-9 M). Antibody 72.38antagonized 12'I-TiTXy binding to Na' channels from differ-ent animal species (fish, avian, and mammalian) and fromdifferent tissues (electroplax, brain, heart, and muscle). More-over, 72.38 has been used for immunofluorescence labeling ofNa+ channels in rat sciatic nodes of Ranvier and cultureddorsal root ganglion cells. Electrophysiological experiments onrat muscle cells fully confirmed the similarity between TiTXYand 72.38 seen in binding experiments. Both produce slowoscillations of the membrane potential accompanied by burstsof action potentials which are due to a selective action on theNa+ channel. TiTXy and 72.38 are without effect on the ionselectivity of the Na+ channel, but they both drastically changethe voltage-dependence of activation and inactivation of theNa+ channel.

Electrophysiological studies have identified a number ofclasses of specific neurotoxins that interact with the voltage-sensitive Na+ channel at discrete receptor sites (1-3): (i)tetrodotoxin (TTX) and saxitoxin (STX), two toxins thatblock Na+ entry through the Na+ channel; (it) the lipophilictoxins such as veratridine and batrachotoxin, which stabilizea permanently open form of the Na+ channel; (iii) pyr-ethroids, a class of highly active insecticides, which prolongthe life time of the open form of the Na+ channel; (iv)polypeptide toxins from North African and North Americanscorpions and from sea anemones, which slow down theinactivation process of the Na+ channel; and (v) one of themost recently discovered classes of toxin, from South andCentral American scorpions (4-6). A very potent repre-sentative of this class is toxin y from Tityus serrulatus(TiTXy), which produces repetitive firing in neuroblastomacells due to a shift in Na+ channel voltage-dependent activa-tion to more negative potentials (7, 8).TTX (9), STX (10, 11), and TiTXy (8, 12, 13) have been

used recently in purification studies of voltage-dependentNa+ channels from different origins. Some of the Na+channel toxins have also been used for the mapping offunctional sites of the channel in native excitable mem-branes, using fluorescence techniques (14).The recent use of monoclonal antibodies (mAbs) against

neurotransmitter receptor molecules has brought spectacu-lar results (see for example ref. 15). The preparation and use

of polyclonal (16) and monoclonal (17) antibodies directedagainst the Na+ channel have already been described; someof them have been reported to block nerve impulse (18).

In this report, we describe the properties of a mAb thatwas generated against a membrane fraction from Electro-phorus electricus electroplax and that has properties likethose of a scorpion neurotoxin. The electrophysiologicalproperties of its action on the Na+ channel are analyzed byusing the voltage-clamp technique.

MATERIALS AND METHODSPure TiTXy from T. serrulatus venom (19) was a gift fromJ. R. Giglio. The preparation of 1251-labeled TiTXy (125I_TiTXy) has been described in detail (6). The [3H]ethyl-enediamine derivative of TTX, [3H]en-TTXI (25 Ci/mmol; 1Ci = 37 GBq), was synthesized according to Chicheporticheet al. (20).

Immunization, Hybridization, and Cell Processing. Micewere immunized with an eel electroplax membrane prepara-tion (4.3 pmol of TTX binding sites/mg of protein) and theirspleen cells were fused with the nonproducing myeloma lineNSO/1 for mAb production as detailed by Meiri et al. (18).Of 520 hybridomas obtained, 7 different clones producingrelevant mAb, as determined by solid phase radioimmuno-assay in the presence or absence of specific neurotoxins forthe Na+ channel (18), were tested for their ability to affectthe binding of some of these neurotoxins to rat brainmembranes. The neurotoxins used were the iodinated de-rivatives of toxin II from the North African scorpionAndroctonus australis (125I-AaH11), toxin V from the seaanemone Anemonia sulcata (125I-ASv), and toxin y from theBrazilian scorpion T. serrulatus (125I-TiTXy) and the triti-ated derivative of tetrodotoxin ([3H]en-TTXI). One clone,SC-72.38, was selected.Assay of l25I-TiTXy Receptor. Specific binding of 125i-

TiTXy to the membrane-bound or solubilized receptor wasmeasured under equilibrium conditions as described (6, 8).Immunoblot Assay. Eighty microliters of P3L (a crude

denatured membrane preparation containing 5 mg ofprotein/ml and 2.3 pmol of 125I-TiTXy binding sites/mg ofprotein) or 400 ,ul of partially purified Na+ channel from ratbrain [wheat germ agglutinin step (8); 0.12 mg of protein/mland 200 pmol of 1251-TiTXy binding sites/mg of protein] weredenatured and subjected to NaDodSO4/PAGE (21). Proteinswere transferred to nitrocellulose, using standard proce-dures (22). After protein transfer, 8-mm nitrocellulose stripswere incubated first with the saturating buffer (150 mMNaCl/10 mM TrisCl, pH 7.4/3% bovine serum albumin/10%

Abbreviations: TiTXy, toxin y from the Brazilian scorpion Tityusserrulatus; TTX, tetrodotoxin; STX, saxitoxin; AaH11, toxin II fromthe North African scorpion Androctonus australis; ASv, toxin Vfrom the sea anemone Anemonia sulcata; 125I., I25I-labeled; mAb,monoclonal antibody; Bmax, maximum binding capacity; DRG,dorsal root ganglion.

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fetal calf serum) for 2 hr at room temperature then overnightat 40C with mAb (150 gg/ml) in the same buffer. The blotswere washed five times (10-min intervals) with 150 mMNaCl/10 mM Tris Cl, pH 7.4/0.1% Tween 20 and thenincubated with affinity-purified 125I-labeled goat anti-mouseIg (106 cpm/ml) for 1 hr at room temperature. Blots thenwere washed again as above, dried, and autoradiographed.

Immunofluorescence Staining. Single myelinated nerve fi-bers were isolated from the sciatic nerve of albino rats andmounted into the chamber used for voltage-clamp. The nodewas externally perfused at 1-2 ml/min with Ringer solution(154 mM NaCl/5.6 mM KCI/1.6 mM CaCl2/20 mM Tris Cl,pH 7.4) at 150C. After 20 min of equilibration, the node wasperfused first for 30 min with 10% horse serum in Ringersolution with or without 10-5 M TiTXy followed by perfu-sion with mAb (50 umg/ml) in 10% horse serum/Ringersolution for 30 min. After a 30-min wash with Ringersolution, the nerve was perfused for 30 min with rhodamine-labeled rabbit-anti mouse IgG (Dakopatts, Glostrup, Den-mark) at 20 ,g/ml, washed again, and then observed in 90%glycerol/Ringer solution under the fluorescence microscopein the experimental chamber. Control incubations were donewith nonrelevant antibodies or with mAb 72.38 that had beenboiled for 10 min. For cultured dorsal root ganglion (DRG)cells of newborn rats, the immunofluorescence staining wasperformed after 5 days in culture, using the same procedureas for the nerve except that inhibition of the staining wasobtained with 10-7 M TiTXy.

Electrophysiological Experiments. Intracellular recordingswere obtained at room temperature (20-22°C) from primarycultures of thigh muscle of newborn rats [myotubes andmyosacs as described (23)]. Voltage-clamp experiments onrat myosacs were performed using a suction-pipette method(7). For K+-current measurements, the ionic composition ofthe solution used for the internal perfusion was 115 mMpotassium glutamate/10 mM Hepes/NaOH. This internalmedium was buffered to pH 7.1 with L-glutamic acid and theosmotic pressure was adjusted to 295 mosM with sucrose.For Na+-current measurements, the external solution con-tained 120 mM NaCl, 25 mM tetraethylammonium chloride,1.8 mM CaC12, and 0.4 mM MgCl2 and was buffered at pH7.4 with 25 mM Hepes/tetramethylammonium hydroxide; itsosmolarity was adjusted to 305 mosM with tetramethylam-monium chloride. The internal perfusion medium contained120 mM CsF and 10 mM NaOH and was buffered to pH 7.4with 25 mM Hepes/L-glutamic acid; the osmotic pressurewas adjusted to 295 mosM with sucrose.

RESULTSBinding Experiments. The binding of [3H]en-TTXI, 125I[

ASv, or 125I-AaH11 to Na+ channels in brain membranes wasrelatively unaffected by high concentrations of 72.38, themAb produced by the clone SC-72.38 (Fig. LA Inset).Conversely, 125I-TiTXy binding was totally inhibited by72.38 at low concentrations. Half-maximum inhibition of125I-TiTXy binding was observed at 5.4 x 10-9 M (Fig. 1A).This apparent K, corresponds to 0.9 ,ug of 72.38 per ml,assuming a Mr of 165,000 for the mAb (Mr 56,500 and 26,000for the heavy and light chains, respectively, by NaDod-S04/PAGE). Control polyclonal mouse antibodies or othermAbs did not affect the binding of the toxin.

Scatchard plots for the binding of 125I-TiTXy to its recep-tor on the Na+ channel in the presence of different concen-trations of 72.38 are shown in Fig. 1B. In the absence of72.38, the dissociation constant (Kd) for the interaction of125i-TiTXy with its receptor site was 5.5 x 10-12 M and themaximal binding capacity (Bma.) was 2.5 pmol/mg of pro-tein. The B1max value was not modified by increasing concen-trations of 72.38 but the apparent Kd value was lowered. This

100

2 CX50~~~~~~~~~~~~~~~~~~~~~~--

B

1- >

CQ

1 4~~~~~~

1251-TiTXy bound, pmol/mg of protein

FIG. 1. (A) Inhibition by mAb 72.38 of 1251-TiTXy binding tolysed and solubilized P3L. Lysed P3L (12 ,ag of protein/ml) wasincubated in choline medium (6) with various concentrations of72.38 (e) or control antibodies (in) for 1 hr at 22°C. Then 18 pM'251-TiTXy was added and the incubation was continued for 1 hr inthe absence (total binding) or in the presence (nonspecific binding)of 1 nM unlabeled TiTX'y. A value of 100%o binding to lysed P3Lcorresponds to 12 pM 1251-TiTX~y specifically bound. Solubilizedmembranes (310 ,ug of protein/ml) were preincubated in assaymedium (8) with 72.38 (A) or control antibodies (o) for 2 hr at 2°Cbefore adding 140 pM 1251-TiTXy. A value of 100%o binding tosolubilized P3L corresponds to 120 pM 425I-TiTXy specificallybound. In both cases (lysed and solubilized P3L) the nonspecificbinding was independent of the antibody concentration and repre-sented 15-20%o of the total binding. (Inset) Effect of 72.38 on thebinding of neurotoxins acting on the Na+ channel. mAb 72.38 (1.4uM) was incubated at 22°C with lysed P3L(T1TX) or synaptosomes(ASv andAaHod)for 1 hr before adding the labeled toxin. Effect onTTX binding was measured usingc 3H]en-aTXs (24) and that onpolypeptide toxin binding, by usingt25p-ASv and(25i-AaH1i (25). (B)Direct binding ofl25d-TiTXy to lysed P3L in the presence of differentconcentrations of 72.38. Lysed P3L (4 ug of protein) was incubatedin 1.4 ml of choline medium (6) with various concentrations ofb25d-TiTXy in the absence (total binding) or in the presence (non-specific binding) of 3 nM unlabeled TiTXy. Incubations were carriedout for 2 hr at room temperature in the presence of no mAb (e)or

bindng f ne [mtxib 72.38] th a cane.mb 238(72.38was1.4ncuae at)2.5 wit lysd3.6 n (oT), or5.4nMaptoso(nset

Kapp = Kd (1 +[mj bfor 38), where Kapp is the apparent dissocia-

tion constant of the TiTXytNa-channel complex in the presence ofdifferent concentrations of 72.38, Kd is the true dissociation constantof the complex in the absence of 72.38, andKo is the true inhibitionconstant of 72.38. Using values of Kapp and Kd calculated from datain the main panel, Kapp/Kd is plotted as a function of the concentra-tion of 72.38. This representation is linear, with the slope giving l/K1.

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is a typical result for competitive inhibition. From therepresentation in Fig. 1B Inset, it is easy to calculate a K, of1.5 x 10-9 M for the complex between 72.38 and the Na'channel. Protection experiments have been carried out onother excitable membranes including E. electricus electro-plax and chick and rat heart membranes. The binding TiTXyto the Na' channel in these different preparations fromdifferent animals is totally inhibited by 72.38, indicating thatmAb 72.38 cross-reacts with Na' channels in all thesetissues. In each case, a dose-response curve for the inhibi-tion by 72.38 of '25I-TiTXy binding has been obtained, andthe corresponding Ki values are given in Table 1.

Fig. lA shows that 72.38 also inhibits the binding of125I-TiTXy to the Na' channel solubilized in Triton X-100.The apparent K, found in this case is 3 x 10-8 M. Afteradequate corrections for the experimental conditions (6), thetrue K, was found to be 2 x 10-9 M, very close to the valuefound for native membranes.

Immunoblotting Experiments. Immunoblots were obtainedafter electrophoresis of denatured crude membranes orhighly enriched Na' channel preparations from rat brain. Nolabeling was obtained with 72.38. This result indicates thatthe native structure of the TiTXy binding site on the Na'channel must be preserved to be recognized by 72.38.

Specific Immunofluorescence Staining of Na' Channel inNerve Cells. The ability of 72.38 to stain the Na' channel inrat sciatic nerve and rat DRG cells was demonstrated by anindirect immunofluorescence technique. Fig. 2 shows spe-cific staining of the nodes of Ranvier in the nerve fiber and ofthe surface membrane in isolated DRG cells. Immunofluo-rescence staining was prevented by 10-7 M TiTXy in DRGcells and byl0-5 M TiTXy on the nodes of Ranvier. A higherconcentration was used in the latter case because of thelower affinity of TiTXy for Na' channels in the node(unpublished observation).

Electrophysiological Experiments. The data reported belowinclude electrophysiological investigations of the actions of72.38 and TiTXy on rat muscle cells in culture.

In cultured rat myotubes, the resting membrane potential(about -60 mV) is not significantly affected by the externalapplication of the antibody 72.38 at a concentration of 50 nM(8.3 Ag/ml). The effect of the antibody is clearly seen afterthe membrane has been hyperpolarized by injecting intracel-lularly a constant current. The membrane potential becomesunstable, displaying slow oscillations accompanied by burstsof action potentials (Fig. 3A). When the hyperpolarizingcurrent is increased, the slow oscillations of the membranepotential tend toward square-shaped waves. This abnormalpattern of activity is similar to that obtained after theexternal application of 2 nM TiTXy to the same preparation

Table 1. Comparative affinity of TiTXy andmAb 72.38 for Na+channel from different excitable tissues

Tissue Kd (TiTXy) Ki (72.38)Rat brain 5.5 x 10-12 M 1.5 x 10-9 M*E. electricus electroplax 3 x 10-12 M 1.6 x 10-9 MChick heart 3 x 10-12 M 0.6 x 10-9 MRatheart 10 x 10-12M 8 x10-9M

E. electricus electroplax membranes (6) (B,,. = 1.5 pmol/ml;5,g of protein/ml), chick heart membranes (13) (B..a = 0.5 pmol/ml; 18,ug of protein/ml), or rat heart membranes (26)(B.,, = 0.18pmol/ml; 29,ug of protein/ml) were incubated in choline medium (6)in the presence of various concentrations of 72.38 and a fixedconcentration of 125I-TiTXy as described in the legend of Fig. 1A.The inhibitory constant(K) is calculated from the concentration of72.38 that induces half-maximum inhibition of 1"I-TiTXy, correctedfor the experimental conditions (6).*K, for rat brain membranes is the value found from the slope of theline in Fig.1B Inset.

A - A: sese~~~~~NI- ':irks ,NO1

NCOAlkW_ fit; In ssAla-

c

D

FIG. 2. Immunofluorescence staining of peripheral nerve (A andB) and DRG cells (C and D). Phase-contrast micrographs are at rightand the corresponding fluorescent images are at left. (A and C)Exposure to 72.38 at 50 /ug/ml. (B and D) Exposure to 72.38 afterpretreatment with 10-1 M or 10-7 M TiTXy, respectively.

(Fig. 3B). External application of TTX (10 4M) to theantibody- and TiTXy-treated rat myotubes suppresses boththe slow waves and the spiking activity (data not shown).

Since the effects of 72.38 are blocked by 10 tkM TTX, thespecific target of the mAb on rat myotubes seems to be thefast Na' channel. Indeed, in voltage-clamp experiments inwhich Na' channels were blocked by 10 uM TTX, theantibody 72.38 (50 nM) had no significant effect on theoutward K+ currents (not shown).

Actions of 72.38 and of TiTXy on the Na' channel havebeen studied comparatively by voltage-clamp experiments.The development of 72.38 (Fig. 4A) and of TiTXy effects(Fig. 4B) takes several minutes. In both cases, the amplitudeof the Na' current is decreased at membrane potentialshigher than -40 mV. Conversely, both molecules induce aninward current for membrane potentials lower than -60 mV,whereas no measurable inward current is evoked by adepolarization of the same amplitude in control experiments.The antibody 72.38 and TiTXy both make the Na' inactiva-tion slow and incomplete at the end of the test pulse (30msec) for membrane depolarizations lower than -40 mV,whereas Na' inactivation kinetics remain similar to that ofthe controls for membrane depolarizations higher than about-20mV. TiTXy (10 nM) more effectively removes the Na'inactivation than the antibody 72.38 (50 nM) (Fig. 4).

Fig. 5 A and B show control peak Na+ current as afunction of membrane potential as well as peak Na+ currentsin the presence of 50 nM 72.38 (Fig. 5A) or 10 nM TiTXy(Fig.SB). A comparison of the two families of peak currentvs. membrane potential curves clearly shows that the peakNa+ currents are increased at membrane potential below-40 mV in the presence of the toxins, whereas the peak Na+currents are reduced at more positive potentials. The Na+current reversal potential (+60 mV) is not affected by thetwo toxins, indicating that they do not modify the ionicselectivity of the Na+ channel. The membrane-potentialdependence of the steady-state inactivation is also affectedby the two toxins (Fig. 5 C and D). Steady-state inactivationcurves were determined using a conventional double-pulse

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A

E0N

2sec

FIG. 3. Spontaneous patternsof burst activity induced by theantibody 72.38 (8.3 ,ug/ml) (A)and by TiTX-y (2 nM) (B) in ratmyotubes. The membrane was

progressively hyperpolarized by acurrent of increasing intensity. (ALeft) I = 1 nA. (A Right) I = 5 nA.(B Left) I from 0.5 to 2 nA. (BRight) I = 5 nA.

protocol. The 10-msec test pulse to +20 mV was keptconstant but the 100-msec pre-pulse was varied from theholding potential (-100 mV) towards hyperpolarized anddepolarized levels. In control experiments, the membranepotential value at which 50% of the Na' current is in-activated (Eh) is -60 ± 5 mV. The antibody 72.38 induces ashift of Eh toward more negative potentials (by -15 mV),whereas the shift for TiTXy was -25 mV.Na+ currents that remain after treatment with 72.38 or

TiTX-y as well as inward currents induced at low membranepotentials (less than -60 mV) are hardly affected by 10 nMTTX. These currents disappear in parallel after the applica-tion of 10 4M TTX. These observations indicate that bothcurrent components have the same TTX sensitivity as nor-

mal fast Na+ currents in rat muscle cells in culture, which

A .:-v

are known to be TTX-resistant (27). The effect of 72.38 isslowly reversible upon washing (>30 min). Conversely, theeffects of TiTXy (2 nM) cannot be reversed even after aprolonged washing (>40 min) (not shown).

DISCUSSIONThe strategy used in this work was to screen for mAbs thatinterfere with the binding of the different neurotoxins knownto be specific for the Na' channel. We obtained a clonesecreting a mAb, 72.38, which competes with a polypeptidetoxin (TiTXy) from the venom of the Brazilian scorpion T.serrulatus for association to the Na' channel present inmembranes from different tissues and animal species (Table1), including electroplax, rat brain, rat and chick hearts, andrat muscle. The data in Fig. 1B show that 72.38 and TiTXy

e.O,/

.w -

FIG. 4. Voltage-clamp analysis of the effects of 72.38 (8.3 /Lg/ml) (A) and TiTXy (2 nM) (B) on rat myosac Na+ currents. Shown are familiesof Na' currents associated with depolarizing voltage steps ranging from 20 mV to 190 mV, with 10-mV step increments, from a holding potentialof -100 mV. (Left) Control. (Center) After a 5-min exposure to the agents. (Right) After a 10-min exposure.

B

ft.

w--

__Ip-

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A

C D

1.01

0.5

-110 -60 mV

1.0 -1

0.5

o-110 -60 mV

FIG. 5. Membrane-potential dependence of the effects of 72.38and TiTXy on peak Na+ current. (A and B) Current (I) vs. potential(V) curves recorded before (e), 5 min after (E), and 10 min after (n)the external application of 72.38 (8.3 ,ug/ml) (A) or TiTXy (1 nM)(B). (C and D) Membrane-potential dependence of steady-state Na'inactivation. The curves show the inactivation of the Na' current(arbitrary units) in the control experiments (e) and the inactivationof the remaining Na' current 10 min after the external application(A) of 72.38 (8.3 ,ug/ml) (C) and TiTXy (1 nM) (D).

share the same binding site on the Na' channel; the affinityof 72.38 for the channel is relatively high, corresponding to aKd of 1.5 x 10-9 M. As for TiTXy, the affinity of 72.38 forthe Na' channel is preserved after solubilization of thechannel. This observation further demonstrates a directinteraction of 72.38 with the Na' channel.

In immunoblotting experiments, 72.38 was unable to labeleither protein present in the rat brain membrane prepara-tion or the purified Na' channel protein. The most prob-able conclusion of this result is that the integrity of thetoxin-binding site must be preserved for the association of72.38. Under conditions of the immunoblotting experiments,TiTXy itself does not recognize the 270,000 Mr proteinknown to be the Na' channel (8, 28). When the integrity ofthe Na' channel is preserved, such as in a node of Ranvierof rat sciatic nerve or in DRG cells, the antibody clearlylabels the channel (Fig. 2).

Since 72.38 binds to the TiTXy binding site on the Na'channel structure, it could act either as an antagonist or anagonist of the scorpion toxin. Electrophysiological experi-ments clearly showed that the second possibility is thecorrect one. Both the immunotoxin and the scorpion toxinchange the voltage-dependence of the activation of the Na'channel, inducing an inward current for membrane poten-tials lower than -60 mV. Both molecules alter the mem-

brane-potential dependence of the steady-state inactivation.Electrophysiological experiments indicate that differences ofeffects in the action of 72.38 and TiTXy on the Na' channelare small.

Polypeptide toxins specific for the Na' channel haveplayed an important role in the analysis of the structural and

functional properties of this channel. One of the mostinteresting of these toxins is certainly TiTXy itself, becauseof its very high affinity for the Na' channels from differentssources and also because, unlike other scorpion toxins(Androctonus, Leiurus, etc.) (25), its binding to Na' chan-nels persists for either depolarized membrane preparationsor solubilized Na' channels (6, 8). The mAb 72.38 willprobably turn out to be another very useful tool, comple-mentary to the small polypeptide toxins, in future studiesconcerning purification of Na' channels from differentsources, immunocytological localization of Na' channels,mobility of the Na' channel protein within the membrane,differentiation of Na' channels, and physio-pathologicalsituations involving Na' channels.

We are grateful to Dr. H. P. M. Vijverberg and Prof. J. Schles-singer for fruitful discussions, to Prof. J. R. Giglio for providingTiTXy, and to C. Roulinat-Bettelheim for expert secretarial assist-ance. This work was supported by grants from the Centre Nationalde la Recherche Scientifique, the Ministbre de l'Industrie et de laRecherche (Grant 83.C.0918), the Association des Myopathes deFrance, and the Schrieber Foundation for Medical Research. H.M.is a Bat Sheva Scholar and a recipient of a short-term EuropeanMolecular Biology Organization fellowship.

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Proc. Natl. Acad. Sci. USA 82 (1985)

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