Extracellular potassium and the regulation of acetylcholine receptor synthesis in embryonic chick muscle cells

Brain Research, 263 (1983) 259-265 Elsevier Biomedical Press

259

Extracellular Potassium and the Regulation of Acetylcholine Receptor Synthesis in Embryonic Chick Muscle Cells BIH-HWA SHIEH, LEO PEZZEMENTI*, and JAKOB SCHMIDT Departments of Pharmacological Sciences and of Biochemistry, State University of New York at Stony Brook, Stony Brook, N Y 11794 (U.S.A.) (Accepted August 17th, 1982) Key words: acetylcholinereceptor-- chick embryo - - myotubes - - regulation - - potassium - - depolarization

The effect of elevated extracellular potassium on acetylcholine receptor synthesis was studied in chick embryonic muscle cultures. At physiological ionic strength, potassium chloride, in the 3.3 to 50 mM range, gave rise to a complex dose-response curve whose prominent features are a considerable reduction of receptor appearance rate at 20 mM and a more than 2-fold increase at higher concentrations. The effect of potassium chloride on receptor synthesis appears to be fairly specific: neither was there a duplication of its effect by other electrolytes or solutes, nor did it alter total protein synthesis or receptor stability by more than 30 Yoat any concentration tested; cellular acetylcholinesterase levels actually declined with increasing KC1 concentrations. In order to explore the mechanism of the potassium effect, tetrodotoxin (10 -8 M), veratridine (3 × 10-8 M), D-600 (1.6 × 10-~ M), and ryanodine (3 x 10-7 M) were tested in the presence of various concentrations of potassium. Sodium channel toxins as well as calcium effectors modified the potassium response. Based on these findings we propose that the effects of potassium are due to: (a) cessation of spontaneous muscle activity upon raising KC1 from 3 to 10 mM; (b) depolarization of the muscle membrane and persistent activation of a calcium channel as concentration is raised from 10 to 20 mM; (c) finally, inactivation or desensitization of the calcium channel, or some other signaling element proximal to the sarcoplasmic reticulum, upon further depolarization. INTRODUCTION N e u r o n a l activity influences m a c r o m o l e c u l a r c o m p o s i t i o n o f effector cells 39. A g o o d e x a m p l e is the v e r t e b r a t e skeletal muscle fiber w h o s e p r o t e i n m e t a b o l i s m is c o n t r o l l e d b y nerve impulse-triggered electrical activity o f the s a r c o l e m m a ; contractile properties, enzymes o f energy m e t a b o l i s m , even the t y p e a n d q u a n t i t y o f m y o s i n i s o p e p t i d e s within a muscle are d e t e r m i n e d b y the electrical stimulus pattern24, 29. M u c h o f w h a t we k n o w a b o u t the c o u p l i n g between m e m b r a n e excitation a n d the r e g u l a t i o n o f p r o t e i n c o n t e n t has been l e a r n e d f r o m e x p e r i m e n t s with c u l t u r e d muscle cells. S p o n t a n e o u s m e m b r a n e activity in such cultures affects the turnover o f m y o s i n 8a a n d the expression o f specific f o r m s o f acetylcholinesterase27, 2s, and, m o s t conspicuously, c o n t r o l s the rate o f acetylcholine re-

c e p t o r synthesis. Electrical s t i m u l a t i o n inhibits rec e p t o r synthesisl0,zl; so do drugs t h a t activate the v o l t a g e - g a t e d s o d i u m channel, e.g. veratridine 5 a n d sea a n e m o n e toxin 4. Conversely, synthesis o f rec e p t o r is e n h a n c e d b y t r e a t m e n t o f cultured muscle cells with t e t r o d o t o x i n which blocks s o d i u m c h a n nels a n d t h e r e b y abolishes s p o n t a n e o u s electrical activityl°,3k T h e question arises if the d e p o l a r i z a tion t h a t a c c o m p a n i e s electrical activity is a necessary link in the signaling p a t h w a y , or p e r h a p s itself suffices to trigger the i n h i b i t i o n o f r e c e p t o r synthesis. Increased extraeellular p o t a s s i u m has been used widely to b r i n g a b o u t a r e d u c t i o n o f the m e m b r a n e potential. T r e a t m e n t o f c u l t u r e d muscle cells with elevated p o t a s s i u m does n o t i m p a i r growth, develo p m e n t a n d fusion, n o r does it p e r m a n e n t l y change m e m b r a n e properties" F i b e r s g r o w n in 25 m M p o t a s -

* Present address: Department of Biology, Franklin and Marshall College, Lancaster, PA 17604, U.S.A. 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

260 sium for prolonged periods of time, when analyzed in 5 mM (normal) potassium, generate overshooting action potentials, and exhibit delayed rectification3Z; acetylcholine receptor clustering is little affected in 25 mM aa or 30 mM KC17. Therefore, elevated potassium was chosen to analyze the regulatory consequences of membrane depolarization. We present here results of a quantitative analysis of the effect of potassium on receptor synthesis in cultured chick myotubes. MATERIALSAND METHODS

Cell culture Primary chick muscle cultures were grown as described previously25. Briefly, cells were obtained from the legs of 12-day embryos, plated in 35 mm gelatin-coated tissue culture dishes at a density of 5 × 105 cells per plate, and cultured in 88 ~o Dulbecco's modified Eagle's medium, 10 ~ horse serum, and 2 ~ chick embryo extract (standard medium) at 37 °C, in a 95 ~o air, 5 ~ CO2, water-saturated atmosphere. To calculate potassium concentrations, horse serum as well as embryo extract were assumed to contain 5 mM KCI. Cultures were treated on day 2 or 3 for 48 h with cytosine arabinoside to suppress proliferation of fibroblasts. Acetylcholine receptor assay a-Bungarotoxin was purified from Bungarus multicintus venom, labeled with 1251, and the monoiodinated derivative isolated as described previously37. The specific activity of this toxin varied from 3.7 × 107 to 1.1 × 107 GBq/mol, depending on the age of the preparation. In some instances, the diiodinated species was used (specific activity from 5.0 to 1.5 × 107 GBq/mol). Cell surface acetylcholine receptors were measured by incubation of cells with 10-8 M [125I]a-bungarotoxin in Hanks' balanced salt solution, containing 1 mg/ml of bovine serum albumin, pH 7.4, for 60 min at 37 °C. The cells were then washed 3 times with phosphate-buffered saline, pH 7.4, solubilized in 0.1 N NaOH, 0.1 ~ SDS, and counted in a gamma counter. Non-specific binding was determined by incubation of cultures in the presence of 10-5 M decamethonium. Receptor appearance rate was assayed by incubating the cells with 10-7 a-bungarotoxin for 1 h, washing thor-

oughly to remove unbound toxin, incubating for 24 additional h, and then assaying for cell surface toxin binding activity as described above. Appearance rate in standard medium varied from 25 to 50 fmol receptor per plate and day. To investigate the effect of variations in the compos'tion of the medium, 6day-old cultures were subjected to the desired treatment (KC1, drug) for 48 h, and receptors appearing during the second 24-h period were measured. Degradation of acetylcholine receptors was determined by the method of Devreotes and Fambrough 11. Briefly, cultures were labeled with [125I]a-bungarotoxin, washed free of excess ligand, then fresh medium of the desired composition was added, and the cultures were assayed for release of radioactivity over a period of 2 days.

Other biochemical assays Total protein was determined by the method of Lowry et al. 20, using bovine serum albumin as a standard. To determine net protein synthetic rate, cultures were treated with the desired medium for one day, then fresh medium and 1.1 × 105 Bq of S-4,5-[3H]leucine (2.4 GBq/mol, Amersham) were added, and incorporation of radioactive label allowed to proceed for another 24 h. Tritiated protein was measured as described by Oh ~1. For determination of total cellular acetylcholinesterase, cultures were washed 3 times with phosphate-buffered saline, pH 7.4 and solubilized in 10 mM sodium phosphate, pH 7.4, 1.0~ Triton X-100. Acetylthiocholine hydrolysis was then measured by the procedure of Ellman et al. 12 in the presence of 10-4 M tetraisopropyl pyrophosphoramide to block non-specific esterases. Chemicals Ryanodine was obtained from Penick, Lyndhurst, N. J., and D-600 from Knoll Pharmaceuticals, Ludwigshafen, Germany. Veratridine and tetrodotoxin were products of Sigma. RESULTS

Effect of potassium on receptor synthesis rate Experiments were carried out by adding, to the culture medium, one half volume of 150 mM NaC1 in which NaC1 was replaced, to varying degrees, by KCI to achieve the desired final potassium concen-

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lular protein synthesis. To determine if potassium affects muscle cell protein metabolism in general, S-[SH]leucine incorporation was measured. Little effect of potassium on net protein synthesis was seen, except at 10 mM KC1 where a 20 ~o stimulation was observed; at the same potassium concentration, enhancement of receptor synthesis was found to be over 40 ~ . Cellular protein content was also measured and found to vary little in response to changes of potassium concentration. Finally, cellular acetylcholinesterase was assayed, and its level found to decrease to about half of control as potassium was raised from 3.5 to 20 mM (Fig. 2).

Pharmacological analysis of potassium response Several drugs known to affect receptor synthesis rate were tested in combination with potassium. To obtain a measure of their specificity, their effects on

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Fig. 1. Receptor appearance rate - - potassium chloride dose-response curve. Concentration of KC1 was varied at constant ionic strength as described in the text, and receptor appearance rate measured as outlined in the Methods section. Values represent averages and S.E.M. of 17-20 individual culture dishes, pooled from 11 independent experiments.

tration. In a mixture of 2 volumes medium and 1 volume 150 m M NaC1, receptor appearance rate was found to be 71.5 4- 4.5~o (S.E.M., n = 13) of that measured in undiluted normal medium. When potassium concentration was raised from 3.3 to 10 mM, appearance rate increased, and then dropped upon further increasing KC1 to 20 raM. The receptor appearance rate then surges as KCI rises to 30 and 40 m M , no further increase was seen at 50 mM (Fig. 1). NaCI, NaBr, sucrose or mannitol, when substituted for KCI, were without effect.

Effect of potassium on other cellular activities Since the half-life of the acetylcholine receptor in cultured chick myotubes is about 20 hla, 25, it is difficult to explain a more than two-fold increase over a period of 24 h by an inhibition of receptor turnover alone. Nevertheless we measured receptor degradation rates and found that receptor stability is indeed not greatly affected by variation in potassium concentration. A modulation of receptor synthesis rate could still reflect a non-specific effect on total cel-

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Fig. 2. Effect of potassium chloride on various cellular parameters. Acetylcholine receptor appearance rate (O), acetylcholine receptor half life (A), total cellular protein (A), incorporation of S-[ZH]leucine into TCA precipitable material (E]), and total acetylcholinesterase (@) were measured as a function of KC1 concentration as described in the Methods section, and values normalized to those obtained at 3.3 m M KCI. Results represent means and S.E.M. of at least 3 independent measurements taken on parallel sets of cultures. S.E.M. of protein measurements was 2.5 ~ or less.

262 TABLE I Effects of drugs administered in standard medium

Total cellular protein, and receptor appearance and degradation rates were measured in triplicate as described in Methods. Values are given in % of untreated controls (averages and S.E.M.). Celhdar protein

Veratridine* * (1-3 × 10_6 M) Tetrodotoxin (10 -6 M) Ryanodine (3 X 10-7 M)

Receptor Receptor appearance half-life rate*

118 ± 2

39 ± 0.3

87 ± 1

96 4- 7

171 4. 3

91 4- 1

111 ± 1

31 4" 2

94 d_ 2

106 4, 2

219 4- 1

132 4. 3

D-600

(1.6 x 10-5 M)

* Data taken from Pezzementi and Schmidt 25 ** Protein measured at 1.5, appearance rate at 1.0, and half-life at 3.0 Ftmol. T

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t o t a l p r o t e i n a n d on r e c e p t o r m e t a b o l i s m were a n a l y z e d in s t a n d a r d m e d i u m . As can be seen f r o m T a b l e I, there are i n d e e d p r o n o u n c e d effects on r e c e p t o r a p p e a r a n c e rates; p r o t e i n levels in the cultures as well as r e c e p t o r t u r n o v e r rates a p p e a r m u c h less affected. The results o f p o t a s s i u m - d r u g c o m b i n a t i o n exp e r i m e n t s are presented in Fig. 3. (a) S o d i u m channel toxins: Veratridine, an act i v a t o r o f the v o l t a g e - g a t e d s o d i u m channel, in low p o t a s s i u m caused a significant r e d u c t i o n in r e c e p t o r a p p e a r a n c e rate, a n d a b o l i s h e d the s t i m u l a t i o n seen at 10 m M KC1. N o significant effect o f veratridine was observed at p o t a s s i u m c o n c e n t r a t i o n s o f 20 m M a n d above. T e t r o d o t o x i n , a p o t e n t b l o c k e r o f the s o d i u m channel, d o u b l e d the rate at p o t a s s i u m c o n c e n t r a t i o n s up to 10 m M , b u t exhibited little s t i m u l a t o r y activity at 20 m M a n d above. (b) C a l c i u m effectors: D-600, a b l o c k e r o f voltage-gated calcium channels, was f o u n d to stimulate r e c e p t o r synthesis across the entire p o t a s s i u m conc e n t r a t i o n range tested. R y a n o d i n e , a d r u g a s s u m e d to release calcium f r o m the s a r c o p l a s m i c reticulum, i n h i b i t e d synthesis regardless o f p o t a s s i u m concentration. DISCUSSION

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Fig. 3. Effect of drugs on potassium chloride dose-response curve. Receptor appearance rate was measured, at various KCI concentrations, in the presence ( 0 ) and absence ( ~ ) of 3 x 10-a M veratridine (A); 10-6 M tetrodotoxin (B); 1.6 x 10-5 M D-600 (C), and 3 x 10-7 M ryanodine (D). Data are

M y o f i b e r s g r o w n in culture usually exhibit spont a n e o u s activity, a n d v a r i a t i o n o f the p o t a s s i u m c o n c e n t r a t i o n - - and, consequently, o f m e m b r a n e p o t e n t i a l - - f r o m a p o i n t where all fibers are active to where n o n e are, should lead to an increase in r e c e p t o r proliferation. Powell et al. z6, w o r k i n g with m o u s e muscle cells in culture, observed c o m p l e t e cessation o f electrical activity u p o n raising extracellular p o t a s s i u m f r o m 3 to 12 m M a n d a n increase in r e c e p t o r density as m e a s u r e d b y a u t o r a d i o g r a p h y ; L6 r a t m y o t u b e s d o n o t spike in 25 m M KC133. In chick e m b r y o m y o t u b e s , likewise, an increase in the p o t a s s i u m c o n c e n t r a t i o n f r o m 5.4 to 12 m M a p p e a r s to suppress s p o n t a n e o u s electrical activity 34. W e m a y therefore assume t h a t the slight st±m-

presented as mean and S.E.M. of 2-4 individual measurements of relative receptor appearance rate (experimental divided by control), pooled from two independent experiments.

263 ulation of receptor synthesis, which is observed upon raising potassium concentration from 3 to 10 mM, is due to the silencing of spontaneous electrical activity. This assumption is corroborated by pharmacological analysis. If several steps within a signaling pathway are amenable to pharmacological manipulation, their proximo-distal sequence may be established by analyzing the effect of combinations of drugs. The assumption is that the state of the component situated downstream (or distally) determines the outcome; stimulation or blockade of a pathway should result from the activation or inhibition, respectively, of a given component regardless of whether a proximal element is simultaneously activated or inhibited. Application of this paradigm to experiments, in which potassium was combined with either tetrodotoxin or veratridine, reveals that external potassium has at least two targets, one identical with the sodium channel or 'upstream' from it, and another situated 'downstream'. Tetrodotoxin, a sodium channel blocker 9 stimulates acetylcholine receptor synthesis in low potassium medium which otherwise enhances spontaneous activity and thereby reduces receptor synthesis rate, whereas veratridine, which generates repetitive discharges in the muscle membrane 17 reactivates the sodium channels silenced by depolarization at 10 mM potassium. At potassium concentrations of around 20 mM and above, on the other hand, the presence of neither inhibitor nor activator of sodium channels seems to matter. The tetrodotoxin experiment indicates that in a muscle fiber that does not spike potassium activates, and at higher concentration inactivates, a signaling pathway component which is located downstream from the action potential channels. These effects are abolished in the presence of D-600, a drug known to block voltage-gated calcium channels in various tissues 14. It has long been known that calcium enters skeletal muscle during electrical stimulation, as well as during potassium contracture 6. A slow calcium current that is blocked by D-600 has recently been observed in frog skeletal muscle2Z,z0. The physiological significance of this current is obscure, as it is too slow to participate in excitationcontraction couplingS,8; in fact, skeletal muscle continues to contract in calcium-free media and in the presence of D-600L In embryonic muscle the situa-

tion is different: action potentials of young embryonic chick myofiber are carried at least in part by calcium currents15,16,z2. Similar observations have been made with rodent myotubes18. We propose that in chick myotubes, depolarization by external potassium leads to steady-state activation of a calcium channel and that the resulting influx of calcium is sufficient to counter - - to some extent at 10 mM, and completely at 20 mM - - the effect of the elimination of spontaneous electrical activity, as indicated by the reduced receptor appearance rate. Further depolarization, brought about by higher potassium concentrations, then causes inactivation or desensitization of the channel or a signaling element located somewhere 'upstream' from the sarcoplasmic reticulum. It may be argued that stimulation of receptor synthesis by elevated extracellular potassium is mediated by an increase in intracellular potassium rather than by membrane depolarization. This is unlikely, however, since treatment with ouabain, which blocks Na ÷, K÷-ATPase and thereby leads to a depolarization accompanied by a decrease in intracellular potassium, stimulates receptor synthesis38. Changes in membrane potential affect the macromolecular composition of other cells. For example, Bandman and Strohman recently reported that elevated (12 mM) KC1 increases the degradation rate of myosin heavy chain polypeptidesz. An interesting parallel to cultured muscle cells is provided by cultured sympathetic ganglion neurons. It is known that these cells, paradoxically, develop cholinergic properties when exposed to a variety of cells or to medium conditioned by such cells; in vivo membrane activity caused by preganglionic stimulation may be required to maintain the adrenergic phenotype of these cells in the face of cholinergic cues released by effector organs ~z. Walicke et al. z5 showed that in vitro, stimulation of nerve activity by 20 mM potassium chloride (and similarly, 1.5 × 10-6 M veratridine or electrical stimulation) was sufficient to prevent the induction of cholinergic properties. They also observed that the effect of potassium could be overcome by the calcium antagonists D-600 and magnesium and concluded that 'depolarization, either steady or accompanying activity, is one of the factors determining whether cultured

264 sympathetic n e u r o n s b e c o m e adrenergic or choliner-

o p p o r t u n i t y to test the p r o p o s i t i o n that m e m b r a n e

gic, a n d this effect m a y be mediated by calcium.' The i n v o l v e m e n t of calcium has since been s u b s t a n t i a t e d

activity leads to altered g e n o m e expression a n d to m o d u l a t i o n of long-term excitability by voltaged e p e n d e n t calcium entry 19.

b y a d d i t i o n a l experiments a6. T h u s b o t h m y o t u b e s a n d sympathetic n e u r o n s can a d o p t either a n 'inactive' (denervated/cholinergic) or a n 'active' (innervated/adrenergic) phenotype, whose expression is

ACKNOWLEDGEMENTS W e t h a n k M a r k Schneider for help with some o f

m e d i a t e d by calcium. Both p r e p a r a t i o n s provide a n

the experiments.

REFERENCES

16 Kano, M. and Yamamoto, M., Development of spike potentials in skeletal muscle cells differentiated ia vitro from chick embryo, J. Cell Physiol., 90 (1977) 439444. 17 Krayer, O. and Acheson, G. H., The pharmacology of the veratrum alkaloids, Physiol. Rev., 26 (1946) 383446. 18 Land, B. R., Sastre, A. and Podleski, T. R., Tetrodotoxin sensitive and insensitive action potentials in myotubes, J. Cell Physiol., 82 (1973) 497-510. 19 Llinas, R., The role of calcium in neuronal function. In Schmitt, F. O. and Worden, F., (Eds.), The Neurosciences, Fourth Study Program, Rockefeller Univ. Press, New York, 1979, pp. 555-571. 20 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-272. 21 Oh, T. H., Neurotrophic effects of sciatic nerve extracts on muscle development in culture, Exp. Neurol., 50 (1976) 376-386. 22 Palade, P. T. and Almers, W., Slow Na and Ca currents across the membrane of frog skeletal muscle fibers, Biophys. J., 21 (1978) 168. 23 Patterson, P. H., Environmental determination of autonomic transmitter functions, Ann. Rev. Neurosci., 1 (1978) 1-17. 24 Pette, D., Muller, W., Leisner, E. and Vrbova, G., Time dependent effects of contractile properties, fiber population, myosin light chains and enzymes of energy metabolism in intermittently and continuously stimulated fast twitch muscles of the rabbit, Pfliigers Arch. Europ. J. Physiol., 364 (1976) 103-112. 25 Pezzementi, L. and Schmidt, J., Ryanodine alters the rate of acetylcholine receptor synthesis in chick skeletal muscle cell cultures, J. Biol. Chem., 256 (1981) 1265112654. 26 Powell, J. A., Friedman, B. A. and Cossi, A., Tissue culture study of murine muscular dysgenesis: role of spontaneous action potential generation in the regulation of muscle maturation, Ann. N.Y. Acad. Sci., 317 (1979) 550-571. 27 Rieger, F., Koenig, J. and Vigny, M., Spontaneous contractile activity and the presence of the 16S form of acetylcholinesterase in rat muscle cells in culture: reversible suppressive action of tetrodotoxin, Develop. BioL, 76 (1980) 358-365. 28 Rubin, L. L., Schuetze, S. M., Weill, C. I. and Fischbach, G. D., Regulation of acetylcholinesterase appearance at neuro-muscular junctions in vitro, Nature (Lond.), 283 (1980) 264-267. 29 Salmons, S. and Sreter, F. A., Significance of impulse activity in the transformation of skeletal muscle type,

1 Almers, W. and Palade, P. T., Slow calcium and potassium currents across frog muscle membrane: measurements with a vaseline-gap technique, J. PhysioL (Lond.), 312 (1981) 159-176. 2 Andersson, K. E., Effects of calcium and calcium antagonists on the excitation-contraction coupling in striated and smooth muscle, Acta pharmacol, toxicoL, 43, Suppl. 1 (1979) 5-14. 3 Bandman, E. and Strohman, R. C., Increased potassium ion inhibits spontaneous contractions and reduces myosin accumulation in cultured chick myotubes, J. Cell Biol., 93 (1982) 698-704. 4 Betz, H., Effects of drug-induced paralysis and depolarization on acetylcholine receptor and cyclic nucleotide levels of chick muscle cultures, FEBS Lett., 118 (1980) 289-292. 5 Betz, H. and Changeux, J. P., Regulation of muscle acetylcholine receptor synthesis in vitro by cyclic nucleotide derivatives, Nature, (Lond.), 278 (1979) 749-752. 6 Bianchi, C. P. and Shanes, A. M., Calcium influx in skeletal muscle at rest, during activity, and during potassium contracture, J. gen. Physiol., 42 (1959) 803-815. 7 Bloch, R. J., Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism, J. Cell Biol., 82 (1979) 6264543. 8 Caputo, C., Nickel substitution for calcium and the time course of potassium contractures of single muscle fibers, J. Muscle Res. Cell Motil., 2 (1981) 167-182. 9 Catterall, W. A., Neurotoxins that act on voltagesensitive sodium channels in excitable membranes, Ann. Rev. Pharmacol. Toxicol., 20 (1980) 15-43. 10 Cohen, S. A. and Fischbach, G. D., Regulation of muscle acetylcholine sensitivity by muscle activity in culture, Science, 181 (1973) 76-78. 11 Devreotes, P. N. and Fambrough, D. M., Acetylcholine receptor turnover in membranes of developing muscle fibers, J. Cell BioL, 65 (1975) 335-358. 12 Ellman, G. L., Courtney, D., Andres, V. and Featherstone, R. M., A rapid colorimetric determination of acetylcholinesterase activity, Biochem. PharmacoL, 7 (1961) 88-95. 13 Fambrough, D. M., Control of acetylcholine receptors in skeletal muscle, Physiol. Rev., 59 (1979) 165-227. 14 Hagiwara, S. and Byerly, L., Calcium channel, Ann. Rev. NeuroscL, 4 (1981) 69-125. 15 Kano, M., Development of excitability in embryonic chick skeletal muscle ceils, J. Cell PhysioL, 86 (1975) 503-510.

265 Nature (Lond.), 263 (1976) 30-34. 30 Sanchez, J. A. and Stefani, E., Inward calcium current in twitch muscle fibers of the frog, J. Physiol. (Lond.), 283 (1978) 197-209. 31 Shainberg, A. and Burnstein, M., Decrease of acetylcholine receptor synthesis in muscle cultures by electrical stimulation, Nature (Lond.), 264 (1976) 368-369. 32 Spector, I. and Prives, J. M., Development of electrophysiological and biochemical membrane properties during differentiation of embryonic skeletal muscle in culture, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 51665170. 33 Steinbach, J. H., Role of muscle activity in nerve muscle interaction in vitro, Nature (Lond.), 248 (1974) 70-71. 34 Strohman, R. C., Bandman, E. and Walker, C. R., Regulation of myosin accumulation by muscle activity in cell culture, J. Muscle Res. Cell Motil., 2 (1981) 269282.

35 Walicke, P. A., Campenot, R. B. and Patterson, P. H., Determination of transmitter function by neuronal activity, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 57675771. 36 Walicke, P. A. and Patterson, P. H., On the role of Ca 2+ in the transmitter choice made by cultured sympathetic neurons, J. Neurosci., 1 (1981) 343-350. 37 Wang, G. and Schmidt, J., Primary structure and binding properties of iodinated derivatives of a-bungarotoxin, J. Biol. Chem., 255 (1980) 11156-11162. 38 Weidoff, P. M., McNamee, M. G. and Wilson, B. W. Modulation of cholinergic proteins and R N A by ouabain in chick muscle cultures, FEBS Lett., 100 (1979) 389-393. 39 Zigmond, R. E. and Bowers, C. W., Influence of nerve activity on the macromolecular content of neurons and their effector organs, Ann. Rev. Physiol. 43 (1981) 673-687.