Pharmacologic properties of voltage-sensitive sodium channels in chick muscle fibers developing in vitro

DEVELOPMENTAL

78,222-230

BIOLOGY

Pharmacologic

(1980)

Properties of Voltage-Sensitive Chick Muscle Fibers Developing WILLIAM

Department

of Pharmacology,

Received

September

Sodium in Vitro

Channels

in

A. CATTERALL

University

of Washington,

21, 1979; accepted

in revised

Seattle, form

January

Washington

98195

15, 1980

The alkaloids veratridine and batrachotoxin cause persistent activation of voltage-sensitive sodium channels in cultured chick muscle cells. The concentration dependence of activation follows a Langmuir isotherm with Koa (veratridine) = 10 $4 and Ko.5 (batrachotoxin) = 1.5 @%4. Veratridine is a partial agonist in activating sodium channels while batrachotoxin is a full agonist. The polypeptides scorpion toxin and sea anemone toxin II enhance activation of sodium channels by veratridine and batrachotoxin, but do not cause persistent activation themselves. Scorpion toxin (KM = 30 nA4) acts at lower concentration than sea anemone toxin (KU, = 70 1144). The polypeptide toxins both reduce Ko, and increase the fraction of sodium channels activated by the partial agonist veratridine, whereas they reduce Ko.5 without effect on the fraction of sodium channels activated by batrachotoxin. These results are consistent with an allosteric model of toxin action in which the alkaloid toxins bind tightly to active states of sodium channels and shift an existing equilibrium in favor of the activated states while the polypeptide toxins reduce the energy required to cause persistent activation. The action of scorpion toxin is inhibited by depolarization in the range of -55 mV to 0 mV. Sodium channels activated by toxin treatment are inhibited by tetrodotoxin and saxitoxin (K, = 6 t-144). A similar high affinity for tetrodotoxin was observed in newly formed myotubes as soon as toxin-activated sodium channels could be detected. Toxinactivated, tetrodotoxin-insensitive sodium channels were not detected. The pharmacologic properties of sodium channels in cultured chick muscle cells resemble those described in previous studies with neuroblastoma cells in all respects studied, except that scorpion toxin action requires higher concentrations and is more voltage dependent.

1978; Jacques et al., 1978). The binding and action of the polypeptide toxins is voltagedependent (Catterall et al., 1976; Catterall, 1977a; Catterall and Beress, 1978). There is a close correlation between voltage-dependent toxin binding and activation of sodium channels (Catterall, 1979). These toxins provide sensitive probes of the structure, function, and voltage dependence of sodium channels. Myotubes formed in vitro from embryonic chick muscle cells are electrically excitable (Fischbach et al., 1971; Kano et al., 1972). Veratridine increases sodium permeability in cultures containing myotubes (Catterall and Nirenberg, 1973). Voltage clamp studies have described rapidly activated sodium conductance and more slowly activated calcium and chloride conductantes (Fukuda et al., 1976). Sodium-depen-

INTRODUCTION

Voltage-sensitive sodium channels in nerve, muscle, and cultured neuroblastoma cells have three separate receptor sites for neurotoxins (reviewed in Catterall, 1980). Receptor site 1 binds the inhibitors tetrodotoxin and saxitoxin (Ritchie and Rogart, 1977). Receptor site 2 binds the lipid-soluble toxins veratridine, batrachotoxin, aconitine, and grayanotoxin. These toxins cause persistent activation of sodium channels by an allosteric mechanism (Catterall, 1975, 197715). Receptor site 3 binds the polypeptides sea anemone toxin and scorpion toxin. The polypeptide toxins block inactivation of sodium channels (reviewed in Catterall, 1980), and enhance activation of sodium channels by the lipid-soluble toxins (Catterall, 1975, 1977b; Catterall and Beress, 222

0012-1606/60/090222-09$02.00/O Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

WILLIAM

A. CATTERALL

Voltage-Sensitive

dent action potentials, sodium conductance, and the veratridine-induced increase in sodium permeability are all blocked by tetrodotoxin (Kano et al., 1972; Harris and Marshall, 19’73; Fukuda et al., 1976; Catterall and Nirenberg, 1973). These results indicate that embryonic chick muscle cells cultured in vitro have rapidly activated sodium channels which resemble those in nerve. In mammalian muscle, the pharmacologic properties of sodium channels are regulated by innervation (Redfern and Thesleff, 1971) and sodium channels in myotubes newly formed in vitro resemble those in denervated muscle (Kidokoro, 1973; Land et al., 1973; Catterall, 1976a; Stallcup and Chon, 1976). In contrast, in chick, denervation does not cause marked changes in sodium channel properties (Nasledov and Thesleff, 1974; Cullen et al., 1975). In this report, I describe the action of the neurotoxins described above on sodium channels in cultured chick muscle cells. MATERIALS

AND METHODS

Chemicals were obtained from the following sources: “NaCl and [4,5-3H]leucine, New England Nuclear; veratridine, Aldrich; scorpion venom (Leiurus quinquestratus) and ouabain, Sigma; DMEM (the Dulbecco-Vogt modification of Eagle’s minimum essential medium), fetal calf serum, and horse serum, Grand Island Biologicals; 1X crystalline trypsin, Worthington Biochemicals; and tetrodotoxin and calf-skin collagen, Calbiochem. Batrachotoxin was provided by Drs. John Daly and Bernhard Witkop, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014. Sea anemone toxin II from Anemonia sulcata was provided by Dr. Laszlo Beress, University of Kiel, Kiel, West Germany. Scorpion toxin was purified as described previously (Catterall, 1976b). Chick embryo extract was prepared as described by Cahn et al. (1967).

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Muscle cell cultures. Suspensions of single muscle cells from thigh muscle of llday-old chick embryos were prepared essentially as described by Fischbach (1972). Cells were seeded at 40,000 cells/cm’ in collagen-coated multiwell plates (Costar, 1.6~cm-diameter wells) in 91% DMEM, 5% horse serum (heat inactivated at 56°C for 30 min), 2% fetal calf serum, 2% chick embryo extract, 50 units/ml streptomycin, and 10 pg/ml penicillin and incubated at 36.5”C in a humidified atmosphere of 10% CO/ 90% air. Cultures were routinely studied after 7 to 12 days in vitro. Fusion into multinucleate myotubes was complete by Day 4 in vitro. In order to prevent overgrowth of the cultures by fibroblasts, all cultures were treated with 10 fl D-arabinofuranosylcytosine for 24 hr beginning on Day 3. The growth medium of all cultures was supplemented with 0.2 $X/ml [“Hlleutine 48 hr prior to experiments to allow use of 3H cpm to normalize protein recovery in uptake experiments. “Na+ Uptake measurements. The increase in sodium permeability induced by neurotoxins which cause persistent activation of sodium channels was measured as described previously for neuroblastoma cells (Catterall, 1976b). Before initiating the uptake experiments, all cell cultures were incubated with 200 nM a-bungarotoxin for 15 min to block acetylcholine receptors. Neurotoxins acting on sodium channels were allowed to equilibrate with their sites of action in a preliminary incubation of 30 min at 36°C in sodium-free medium consisting of 130 mM choline chloride, 50 mM Hepes (adjusted to pH 7.4 with Tris base), 5.5 mM glucose, 0.8 mM MgS04, 5.4 mM KCl, and 1 mg/ml bovine serum albumin. Under these conditions influx of Na’ during the preliminary incubation is prevented. In previous experiments with neuroblastoma cells, loss of response to neurotoxins was observed during prolonged incubation under conditions where all the sodium channels in the cells were activated. This was correlated with loss of intracellular K’

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224

(Catterall, 1976b). In contrast, no loss of responsiveness to neurotoxins was observed in primary chick muscle cultures indicating that these cells are less leaky than neuroblastoma cells. Preliminary incubation in sodium-free, choline-substituted medium was, therefore, adequate to prevent ionic rearrangements. After equilibration with toxins, the initial rate of 22Na+ uptake was determined in medium consisting of 10 m&f NaCl, 120 n&f choline chloride, 50 mA4 Hepes (adjusted to pH 7.4 with Tris base), 5.5 mM glucose, 5 mM ouabain, 0.8 mM MgSO+ and 5.4 mM KCl. Uptake of 22Na+ was terminated by washing three times with 3 ml of wash medium consisting of 163 mll4 choline chloride, 5 rnJ4 Hepes (adjusted to pH 7.4 with Tris base), 1.8 mM CaC12, and 0.8 mM MgS04. Cells were suspended in 0.4 M NaOH and radioactivity was determined as described previously (Catterall, 1976b). Cell protein was determined by the method of Lowry et al. (1951). The increase in sodium permeability caused by neurotoxins is presented in nmole - min-’ . mg-‘. RESULTS

Preliminary studies showed that sodium channels in chick muscle cells were activated by batrachotoxin as well as veratridine and that scorpion toxin and sea anemone toxin II enhanced activation by both lipid-soluble toxins. Figure 1 illustrates the effect of incubation with batrachotoxin plus scorpion toxin on the initial rate of passive sodium influx. Toxin treatment causes a sixfold increase in the initial rate of uptake. The rate of influx remains approximately linear for 1 min under these conditions. A standard assay time of 30 set was used in subsequent experiments. The fraction of sodium channels activated by neurotoxins is directly proportional to the increase in sodium permeability (PNJ. It is important to verify that the “Na+ influx measured is linearly proportional to sodium permeability. The unidirectional sodium influx is proportional to

VOLUME ?8,1980

0

.5

TIME

1.0

(min)

15

2.0

FIG. 1. Initial rate of toxin-stimulated “Na+ uptake. Chick muscle cultures were incubated for 20 min at 36°C with no addition (0) or with 300 nit4 scorpion toxin and 200 n&f batrachotoxin (0) in sodium-free medium. The initial rate of “‘Na’ uptake was then measured at 36°C as described under Materials and Methods. sodium permeability if the influx varies linearly with extracellular sodium concentration (Eq. 2.4, Hodgkin and Katz, 1949). Conditions under which this criterion is fulfilled have been described for neuroblastoma cells (Catterall, 1977b). The neuroblastoma cells were incubated with neurotoxins in sodium-free medium to allow equilibration with receptor sites without accumulation of intracellular sodium. Initial rates of 22Na+ influx were then measured in medium of low sodium concentration to prevent depolarization during the flux measurement. This approach (see under Materials and Methods) has also proven adequate with cultured muscle cells. Figure 2 illustrates the dependence of 22Na+ influx rate on extracellular sodium concentration under conditions of maximum sodium channel activation (batrachotoxin plus scorpion toxin). Influx depends linearly on [Na+lout up to 15 n&f Na+. A standard concentration of 10 mM was used in subsequent experiments. Equilibrium concentration-effect curves for veratridine-activation of sodium channels in the presence and absence of scorpion

WILLIAM

A. CATTERALL

Voltage-Sensitive

toxin are illustrated in Fig. 3. In the absence of scorpion toxin (O), veratridine increases 22Na+influx by 22 nmole.min-’ .mg-’ with a JGs of 10 $kf. The concentration-effect curve fits a simple Langmuir isotherm. Scorpion toxin alone has no effect on 22Na+ influx (points on ordinate in Fig. 3). In the presence of scorpion toxin, V,,, for veratridine-dependent “Na+ influx is increased

225

Sodium Channels

to approximately 70 nmole . min-’ .mg-’ and Ko.~ is reduced to approximately 1 $V. Thus, the enhancement of veratridine action by scorpion toxin is due to both an increase in V,,, and a decrease in Ko.5. Equilibrium concentration-effect curves for batrachotoxin activation of sodium channels are illustrated in Fig. 4. In the absence of polypeptide toxins, batrachotoxin activates sodium channels in the range of 0.2 to 2 fl (Fig. 4, top, A). Doublereciprocal plot analysis of those data gives straight lines with V,,, = 143 nmole/min/ mg and Ko.5 = 1.5 fl (Fig. 4, bottom, A). Incubation with sea anemone toxin II (300 n&f) enhances batrachotoxin action (Fig. 4, top, 0) by reducing Ko.5 to 0.22 @4 without altering Vmax significantly (Fig. 4, bottom,

[Na+lou, , mM FIG. 2. Dependence of toxin-stimulated “Na+ uptake on [Na+luu,. Chick muscle cultures were incubated for 30 min at 36°C with 300 m&f scorpion toxin and 200 nM batrachotoxin in sodium-free medium. The initial rate of *‘Na+ uptake was then measured for 30 set at 36°C as described under Materials and Methods.

[BATRACH~T~XINI,

[VERATRIDINE] ,M FIG. 3. Activation of sodium channels by veratridine and scorpion toxin. Chick muscle cultures were incubated for 20 min at 36°C in sodium-free medium with the concentrations of veratridine indicated on the abscissa with (0) or without (0) 100 n&f scorpion toxin. The initial rate of “Na+ uptake was then measured for 30 set at 36°C as described under Materials and Methods.

h4

FIG. 4. Activation of sodium channels by batrachotoxin, scorpion toxin, and sea anemone toxin. Chick muscle cultures were incubated for 20 min at 36°C in sodium-free medium containing the indicated concentrations of batrachotoxin and no additions (A), 300 niI4 scorpion toxin (O), or 300 r&f sea anemone toxin (0). The initial rate of ‘*Na+ uptake was then measured for 30 set at 36°C as described under Materials and Methods.

226

DEVELOPMENTALBIOLOGY

0). Incubation with scorpion toxin is even more effective in enhancing batrachotoxin action (Fig. 4, top, 0). Ko.5 is reduced to 7 nM without a significant alteration in V,,, (Fig. 4, bottom, 0). Thus, both scorpion toxin and sea anemone toxin II enhance batrachotoxin action by reducing Ko.~ without effect on V,,,. These results can be considered in terms of an allosteric model of neurotoxin action described previously in experiments on neuroblastoma cells (Catterall, 1977b). In this model, the activation of the sodium channel by lipid-soluble toxins results from their ability to bind with high affinity to activated states of sodium channels and thereby shift an existing equilibrium between active and inactive states. Polypeptide toxins enhance activation by lipid-soluble toxins by reducing the energy required to make the transition from inactive to active states. In terms of this model, veratridine, aconitine, and grayanotoxin were partial agonists in neuroblastoma cells activating only a fraction of sodium channels at saturation. Batrachotoxin was a full agonist activating all of the sodium channels. Polypeptide toxins both increased the fraction of sodium channels activated at saturation and reduced Ko.5 for the partial agonists. Polypeptide toxins reduced K,x, without effect on the fraction of sodium channels activated by the full agonist batrachotoxin. The affinity changes observed for partial agonists were smaller than for full agonists. The results with chick muscle cells can be interpreted in terms of the same model. Veratridine is a partial agonist activating only a fraction of the sodium channels in cultured muscle cells (Fig. 3). Scorpion toxin and sea anemone toxin increase both the fraction of sodium channels activated at saturation and reduce Ko.5 (Fig. 3). Batrachotoxin is a full agonist (Fig. 4). Scorpion toxin and sea anemone toxin reduce Ko.5 without affecting the fraction of sodium channels activated at saturation (Fig. 4). As

VOLUME 78,198O

required by the model, the affinity increase for the partial agonist veratridine is smaller than for the full agonist batrachotoxin. These results are consistent with the conclusion that sodium channels in cultured chick muscle cells have allosteric interactions between receptor sites for lipid-soluble toxins and for polypeptide toxins that are similar to those previously described for neuroblastoma cells (Catterall, 1977b). The concentration dependence of polypeptide toxin action is illustrated in Fig. 5. The scorpion toxin-dependent increase in “Na+ upV max for veratridine-stimulated

[SCORPION

FIG. 5.

TOXIN],

M

Concentration dependence of scorpion toxin and sea anemone toxin action. (A) Chick muscle cultures were incubated for 20 min at 36°C with the indicated concentrations of scorpion toxin (0) or sea anemone toxin (0) in sodium-free medium. Initial rates of ‘*Na+ uptake were then measured for 30 set at 36°C in the presence of 200 fl veratridine and the same concentrations of scorpion toxin and sea anemone toxin as described under Materials and Methods. (B) Chick muscle cultures were incubated for 20 min at 36°C with the indicated concentrations of scorpion toxin plus 200 m%f batrachotoxin in sodiumfree medium. Initial rates of **Na+ uptake were then measured as described under Materials and Methods.

WILLIAM

A. CATTERALL

Voltage-Sensitive

take (Fig. 5, top) and the scorpion toxindependent decrease in IG.5 for batrachotoxin action (Fig. 5, bottom) both give values of Ko.5= 30 nM for scorpion toxin. In both cases,the concentration-effect curves are fit by simple Langmuir isotherms. These results support the conclusion that both effects are due to interaction of scorpion toxin with the same receptor sites. Sea anemone toxin II is somewhat less potent with Ko.5 = 70 r&f (Fig. 5, top). The binding and action of scorpion toxin are inhibited by depolarization in neuroblastoma cells and frog skeletal muscle (Catterall et al., 1976; Catterall, 1977a, 1979). Since scorpion toxin action is slowly reversed in cultured chick muscle cells (tl,z = 18 min), the effect of membrane potential on toxin binding can be estimated in a twostep experiment. In the first incubation, cells are allowed to bind scorpion toxin in media of increasing K+ concentrations. The cells are then washed to remove unbound toxin and scorpion toxin enhancement of veratridine activation is measured in a second incubation at K’ = 5.4 mM in which “Na+ influx is determined. Thus, the binding of scorpion toxin takes place at different membrane potentials but its effect on veratridine activation of sodium channeIs is measured at the resting membrane potential. Figure 6 illustrates the reduction in scorpion toxin enhancement of veratridine activation. caused by depolarization with extracellular K+. The effect of 300 n&f scorpion toxin is blocked 94% by depolarization with 135 mA4 K’. The resting membrane potential of cultured chick muscle cells has been reported to be approximately -55 mV (Fischbach et al,, 1971; Harris and Marshall, 1973; Ritchie and Fambrough, 1975; Spector and Prives, 1977). Since the cells have a high resting potassium permeability (Ritchie and Fambrough, 1975), the membrane potential should be near 0 mV at 135 mit4 K+. Thus, inhibition of scorpion toxin action occurs during depolarization from -55 mV to near 0 mV in cultured chick

227

Sodium Channels

1 80-

[K+l ,mM

6. Membrane potential dependence of scorpion toxin action. Chick muscle cultures were incubated for 20 min at 36°C with 300 n&f scorpion toxin in sodium-free medium with the K’ concentrations indicated on the abscissa. Choline concentration was adjusted so that [choline’]+[K’] = 135.4 miW. The cultures were then briefly rinsed in medium with 5 m&f K’ and initial rates of “Na+ uptake were measured at 36°C for 30 set in the presence of 200 aM veratridine as described under Materials and Methods. FIG.

muscle cells as observed previously in neuroblastoma cells (Catterall et al., 1976; Catterall, 1977a) and frog skeletal muscle (Catterall, 1979). Half-maximal inhibition of scorpion toxin action was observed at 20 mM K+ (Fig. 6). Resting membrane potentials at different K+ concentrations have been reported for cultured rat muscle cells, but not for chick muscle cells (Ritchie and Fambrough, 1975). Resting membrane potentials are similar for rat and chick cells at K+ = 5.4 mM (Ritchie and Fambrough, 1975). If they are similar at other K’ concentrations, halfmaximal inhibition at 20 mM K+ in welldifferentiated cultures would imply a membrane potential of -44 mV for 50% reduction of toxin action. This value is similar to the potential of -42 mV for 50% inhibition of scorpion toxin binding to sodium channels in frog muscle, but more negative than the value of -28 mV observed in neuroblastoma cells (Catterall, 1979).

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The KD for binding of tetrodotoxin to sodium channels in frog muscle is 3 to 5 nM (Almers and Levinson, 1975; Jaimovich et al., 1976). Sodium channels in cultured chick muscle cells are blocked by tetrodotoxin (Kano et al., 1972; Harris and Marshall, 1973; Fukuda et al., 1976), but concentration-effect relationships have not been reported. Figure 7 illustrates the concentration dependence of inhibition of toxin-activated sodium channels by tetrodotoxin and saxitoxin. Both toxins inhibit 22Na’ influx with a Ki of 6 nM. The inhibition data fit a simple Langmuir isotherm. Thus, chick muscle cells cultured in vitro have sodium channels with high-affinity receptor sites for tetrodotoxin and saxitoxin. In this respect, cultured chick muscle cells differ from cultured rat muscle cells which have only sodium channels with low-affinity receptor sites for tetrodotoxin and saxitoxin (Catterall, 1976a; Stallcup and Cohn, 1976). Previous electrophysiologic studies have

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shown that sodium-dependent action potentials appear after fusion of muscle cells into multinucleated myotubes and that the rate of rise of the action potential (dV/dT) increased progressively up to 8 days in vitro (Spector and Prives, 1977). Consistent with these results, we find that toxin-stimulated 22Na+ influx is first observable after 2 days in vitro as fusion begins and increases progressively over 2 weeks in vitro (Table 1). Since fusion into multinucleate myotubes is complete by Day 4, most of the increase in toxin-stimulated influx occurs in postfusion myotubes. In order to determine whether sodium channels with low-affinity receptor sites for tetrodotoxin appear in early stages of development, the inhibition of 22Na+ influx by 30 nM tetrodotoxin was determined in several groups of cultures as a function of culture age. At the earliest time that toxin-stimulated influx was measurable, 30 nM tetrodotoxin caused 70% inhibition (Table 1). This inhibition increased to 80 to 90% from 6 to 10 days in vitro. Since a KD of 6 & (Fig. 7) implies 83% inhibition at 30 niV, nearly all of the sodium channels in newly formed chick myotubes have high affinity for tetrodotoxin. DISCUSSION

The pharmacologic properties of sodium channels in chick muscle cells cultured in vitro are similar to those previously described for neuroblastoma cells in nearly all TABLE

[TOXIN],

M

FIG. 7. Inhibition of sodium channels by tetrodotoxin and saxitoxin. Chick muscle cultures were incubated with 300 nM scorpion toxin and the indicated concentrations of tetrodotoxin (0) or saxitoxin (01. Initial rates of **Na+ uptake were then measured for 30 set at 36’C in the presence of 200 fl veratridine and the same concentrations of tetrodotoxin and saxitoxin as described under Materials and Methods.

1

EFFECT

OF CULTURE AGE ON TETRODOTOXIN INHIBITION OF SODIUM CHANNELS

Days

in vitro

3 6 8 10

**Na+ Uptake” (nmole. min-’ me’) 4.3 18.1 22.2 38.1

.

Percentage inhibition by 30 n M TTX ” 70 95 81 88

” “Na+ uptake was measured in cultures preincubated with 200 n M batrachotoxin and 300 n M scorpion toxin as described under Materials and Methods. Where indicated, 30 nM tetrodotoxin was present in both preliminary incubation and assay.

WILLIAM A. CATTERALL

Voltage-Sensitive

respects studied. Veratridine is a partial agonist in activating sodium channels while batrachotoxin is a full agonist. Batrachotoxin binds to their common receptor site with higher affinity than veratridine. Both scorpion toxin and sea anemone toxin II enhance activation by veratridine and batrachotoxin. Scorpion toxin is more potent in enhancing activation. The allosteric mechanism described for activation of sodium channels in neuroblastoma cells adequately describes the data on chick muscle cells. The action of scorpion toxin is inhibited by depolarization of the cells. Tetrodotoxin and saxitoxin block sodium channels by binding to a high-affinity receptor site. These common properties suggest great similarity between sodium channels in neuroblastoma and chick muscle cells cultured in vitro. Two differences in scorpion toxin action were noted. The KD for scorpion toxin in chick muscle cells (30 r&f) is 30-fold higher than in neuroblastoma cells (1 nM, Catterall, 1976b) in spite of the more negative membrane potential of muscle cells. The membrane potential at which toxin action is inhibited 50% is more negative in chick muscle cells (-44 mV) than in neuroblastoma cells (-28 mV). Both of these values for chick muscle cells are similar to the Ku and membrane potential for 50% inhibition of binding observed in scorpion toxin binding studies with intact frog muscle [I4 nM and -42 mV, respectively (Catterall, 1979)]. Thus, a lower affinity and greater voltage sensitivity of scorpion toxin binding may be characteristic of sodium channels in muscle. The voltage sensitivity of scorpion toxin binding is of particular interest because our studies of scorpion toxin binding in frog muscle show that it reflects the voltage dependence of sodium channel activation. In voltage clamp studies of chick muscle cells, Fukuda et al. (Fig. 4 of Fukuda et al., 1976) observed activation of sodium currents between -60 and -20 mV with half

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maximal activation at approximately -42 mV. This compares well with the value of -44 mV for 50% reduction of scorpion toxin action suggested by the results presented here. Thus, the results with cultured chick muscle cells are consistent with the conclusion that the voltage dependence of scorpion toxin binding and sodium channel activation are similar in this system, also. The pharmacologic properties of sodium channels in cultured chick muscle cells differ from those in cultured rat muscle cells in two respects. Rat muscle cells in primary culture (unpublished experiments) and in clonal cell lines (Catterall, 1976a; Stallcup and Cohn, 1976) have sodium channels with low-affinity receptor sites for tetrodotoxin and saxitoxin (KD values of approximately 1 w for tetrodotoxin and 0.3 $%f for saxitoxin). Sodium channels in cultured rat muscle cells also have polypeptide toxin receptor sites with higher affinity for sea anemone toxin than scorpion toxin and with voltage-dependent allosteric interactions among sea anemone toxin receptor sites. These differences may reflect differences in the regulation of sodium channel properties in muscle from mammalian and nonmammalian vertebrates. In frog and chick, denervation has no effect on sodium channel properties (Nasledov and Thesleff, 1974; Cullen et al., 1975). In contrast, in rat, mouse, and cat, denervation causes the appearance of tetrodotoxin-resistant sodium channels (Redfern and Thesleff, 1971). Similarly, uninnervated chick muscle cells in culture have tetrodotoxin-sensitive action potentials, while embryonic and cultured rat muscle cells have tetrodotoxin-resistant action potentials (Kidokoro, 1973; Land et al., 1973; Harris and Marshall, 1973) and sodium channels with relatively low affinity for tetrodotoxin (Catterall, 1976a; Stallcup and Cohn, 1976). Thus, differences in sodium channel regulation by innervation in mammalian and nonmammalian vertebrates described in denervation experiments are also observed in newly formed

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myotubes in cell culture. Muscle cells in culture may provide a suitable experimental system in which to examine these regulatory mechanisms in more detail. I thank Mrs. Sherry Pesheck for excellent technical assistance. This work was supported by a grant from the Muscular Dystrophy Association.

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W. A., RAY, R., and MORROW, C. S. (1976). Proc. Nat. Acad. Sci. USA 73,2682-2686. CULLEN, M. J., HARRIS, J. B., MARSHALL, M. W., and WARD, M. R. (1975). J. Physiol. 245,371-385. FISCHBACH, G. D. (1972). Develop. Biol. 28,407-429. FISCHBACH, G., NAMEROFF, M., and NELSON, P. G. (1971). J. Cell Physiol. 78,289-300. FUKUDA, J., FISCHBACH, G. D., and SMITH, T. G. (1976). Develop. Biol. 49,412-424. HARRIS, J. B., and MARSHALL, M. W. (1973). Nature New Biol. 243, 191-192. HODGKIN, A. L., and KATZ, B. (1949). J. Physiol. 108, 37-77. JACQUES, Y., FOSSET, M., and LAZDUNSKI, M. (1978). J. Biol. Chem. 253,7383-7392. JAIMOVICH, E., VENOSA, R. A., SHRAGER, P., and HOROWICZ, P. (1976). J. Gen. Physiol. 67, 399-416. KANO, M., SHIMADA, Y., and ISHIKAWA, K. (1972). J. Cell. Physiol. 79, 363-366. KIDOKORO, Y. (1973). Nature (London) 241, 158-159. LAND, B. R., SASTRE, A., and PODLESKI, T. R. (1973). J. Cell Physiol. 82, 497-510. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). J. Biol. Chem. 193,265-275. NASLEDOV, G. A., and THESLEFF, S. (1974). Acta Physiol. Stand. 90, 370-380. REDFERN, G. A., and THESLEFF, S. (1971). Acta Physiol. Stand. 82, 70-78. RITCHIE, A. K., and FAMBROUGH, D. M. (1975). J. Gen. Physiol. 66, 327-355. RITCHIE, J. M., and ROGART, R. B. (1977) Rev. Physiol. Biochem. Pharmacol. 79, 1-51. SPECTOR, I., and PRIVES, J. M. (1977). Proc. Nat. Acad. Sci. USA 74, 5166-5170. STALLCUP, W., and COHN, M. (1976). Exp. Cell Res. 98,277-284. CATTERALL,