The Journal of March 1966, 6(3): 691-700

Loss of Receptor Clusters Induced by Treatment of Cultured Rat Myotubes with Carbachol

Robert J. Bloch Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Prolonged exposure to carbachol disrupts the acetylcholine re- of carbachol; (2) the cation flux into myotubes following the ceptor (AChR) clusters of cultured rat myotubes without causing conformational change; (3) the depolarization following cation myotube loss. The effect is reversible, and is dependent on tem- flux. I show that the second possibility, cation flux through the perature. Half-maximal cluster loss is achieved at 3 PM car- receptor channel, accounts for the destabilizing effect of car- bachol. Cluster loss is also caused by other agonists of the AChR bachol on AChR clusters. These results are consistent with the and is blocked by receptor antagonists. QX314 (a lidocaine de- idea that the stability of the AChR clusters of cultured rat myo- rivative), meproadifen, and fluphenazine also completely block tubes is dependent on the intracellular ionic milieu. cluster loss caused by carbachol. These results are consistent with the idea that cluster loss caused by carbachol and other Materials and Methods receptor agonists results from their interaction with the AChR, Methods for preparing myotube cultures, for staining them with tetra- and the consequent influx of cations into the myotubes. Several methylrhodamine-cu-bungarotoxin (R-BT; Ravdin and Axelrod, 1977), experiments suggest that extracellular Na+ and Ca*+ are re- and for analyzing AChR clusters have been presented in detail elsewhere quired, and that at least Na+ must permeate the AChR ion chan- (Bloch, 1979, 1983). In brief, rat myotube cultures, grown on glass nel for full cluster loss to occur in the presence of carbachol. coverslips, are washed with Dulbecco-Vogt modified Eagle’s medium (DMEM) buffered with HEPES, then covered with DME&I containing Depolarization alone is not sufficient to cause cluster loss, how- 5% fetal calf (Gibco. Grand Island. NY) or cadet calf serum (Biocell ever. Ca*+-activated proteases do not play a significant role in Laboratories, barson, CA), and incibateh with or without the addition carbachol-induced cluster loss. of drugs for 6 hr at 37°C in an atmosphere of 94% air, 6% CO,. Cultures are washed extensivelv with HEPES-buffered DMEM. stained with R-BT. The clustering of acetylcholine receptors (AChR) at sites of nerve- fixed, and observed-under fluorescence optics to hetermine the per: muscle contact is one of the earliest events in the differentiation centage of the total myotubes displaying large, flat AChR clusters on of the postsynaptic region of the (Fam- their ventral surfaces. brough, 1979). To understand the synaptogenic interactions be- A cluster is defined as a discrete region of the myotube surface, -40 tween nerve and muscle, 1 have been studying AChR clustering, pm2 in area, that stains brightly with R-BT, is neither linear nor an in vivo and in vitro, in aneural cultures of rat myotubes. The artifact of the overlapping of two or more cells, and is not located on a cellular process isolated from the muscle cell or detached from the organization and properties of the AChR clusters in newly in- tissue culture substrate (Bloch. 1979). Usuallv 50 mvotubes ner culture nervated muscle and in cultured myotubes seem to be similar were analyzed, and eacd expehmental value Is the average ok duplicate in the rat (Steinbach and Bloch, in press). One similarity is that or triplicate determinations. Values generally vary by f 10% myotubes AChR clusters in cultured rat myotubes and at the embryonic with clusters (e.g., Tables 3 and 5, below). postsynaptic membrane are susceptible to disruption by pro- Influx experiments with 22Na+ were performed as described by Bloch longed treatment with carbachol, an AChR agonist (Bloch, 1979; (1979) and Stallcup and Cohn (1976), except that the temperature was Bloch and Steinbach, 198 1). To learn more about this phenom- 35-37“C, and incubations were performed for 15 min. Longer exposure enon, I have studied the factors involved in carbachol-induced to carbachol before assay also resulted in increased 22Na+ influx, but AChR cluster loss in cultured cells. was not as quantitatively reproducible and so was not pursued further. The sources of most materials have been reported earlier (Bloch, In the experiments reported here, I show, first, that carbachol 1979). Nicotinic and muscarinic cholinergic drugs and other neuro- does not exert its effect by causing loss or irreversible damage transmitter receptor ligands were purchased from Sigma Chemical Com- to myotubes, and, second, that it destabilizes AChR clusters by pany (St. Louis, MO) or were gifts from Dr. J. Patrick (Neurobiology interacting with the nicotinic receptor. I then consider three Laboratory, The Salk Institute, La Jolla, CA). Leupeptin, aprotinin, actions of carbachol that could destabilize AChR clusters: (1) pepstatin, and L-seryl-L-leucine were purchased from Sigma. The pro- the conformational change induced in the receptor by binding tease inhibitor, Ep475, was kindly provided by Dr. L. Waxman (De- partment of Physiology, Harvard Medical School). Fluphenazine and meproadifen were the gifts of Drs. B. Krueger and E. Albuquerque Received Feb. 27, 1985; revised May 20, 1985; accepted June 11, 1985. (Departments of Physiology, and Pharmacology, respectively, Univer- I thank Dr. Stephen Heinemann, in whose laboratory at the Salk Institute (La sity of Maryland School of Medicine). Jolla, CA) much of this work was performed, for his encouragement and financial support. I also thank Dr. P. Brehm for his measurements of membrane potential, and Drs. J. Patrick, J. H. Steinbach, and R. Gruener for their useful suggestions. Results Drs. D. Burt. L. Waxman. B. Krueaer, and E. X. Albuquerque generously offered their reagents and advice, and Ms. w. tiesneck assisted ih some ofthe experiments. Cluster losscaused by carbachol I began this work as a Fellow of the San Diego Chapter of the American Heart Association, with support from the Samuel Roberts Noble Foundation of Ard- In cultures of rat myotubes, 70-90% of the myotubes normally more, OK, and from grants to Dr. Heinemann from the National Institutes of display large clustersof AChR, which can be visualized after Health (NS 11549) aid the Muscular Dystrophy Association. More recently, I staining with R-BT. Upon exposure to 10m4M carbachol, the have been supported by grants from the National Institutes of Health (NS 17282) and the Muscular Dystrophy Association, as well as by a M&night Scholar’s fraction of myotubes displaying clusters decreasesto ~30% Award and a Research Career Development Award (NS 00679). (Bloch, 1979). This lossof clustersoccurs as a linear function Copyright 0 1986 Society for Neuroscience 0270-6474/86/030691-10$02.00/O of time up to 6 hr (Fig. 1). As the effectsof carbacholon AChR

691 692 Bloch Vol. 6, No. 3, Mar. 1986

Table 1. Effect of carbachol on AChR clusters and number of myotubes

Carbachol No carbachol Experiment Experiment 1 2 Total O/o 1 2 Total % Myotubes t=O 37 52 88 100 56 53 106 100 t=6hr 32 41 73 83 43 50 93 88 Myotubeswith clusters 2 4 6 t=6hr 30 3 4 35 38 73 70 Time (hr) Myotube cultures on glass coverslips were mounted into a special incubation chamber (Anderson et al., 1977). Selected fields were photographed at low Figure 1. The time course of AChR clusterloss in the presenceof magnification, and their grid positions were noted. These photographs subsequently carbachol.Myotube cultureswere incubated in medium(open circles) gave the number of myotubes present in the selected fields at the beginning of the or in mediumcontaining either 10e4M carbachol(crosses) or 10m4M experiment (t = 0). The cultures were then placed into a solution consisting of d-tubocurarine(triangles). At the times indicated,the cultureswere DMEM plus 5% cadet calf serum. Half of the chambers also contained 10m4 M removedfrom the incubator,washed extensively with bufferedmedium, carbachol. Cultures were incubated for 6 hr at 37”C, washed free of the incubation medium and stained with R-BT. After staining, the cultures were fixed in 2% stainedwith R-BT, and observedunder fluorescence.Myotubes with paraformaldehyde. The same grid positions were relocated and photographed. AChR clusterswere quantitated as described in Methods.The results These photographs subsequently gave the number of myotubes present in the showthat clusterloss is induced by carbachol,but not by d-tubocurarine, same fields at the termination of the experiment (t = 6 hr). The myotubes in these and that it proceedsas a linearfunction of time of treatment. selected fields were then observed with fluorescence optics under high magnification to determine whether AChR clusters were present, as outlined in Methods. These data are contained in the row labeled “Myotubeswith clusters,” t = 6 hr. clustersare apparent at 6 hr, this time was routinely usedin the experiments presentedhere. Data in Figure 1 further suggest that the failure to detect AChR clusters was not due to incom- bated for an additional 18 hr. Upon staining with R-BT, most plete removal of carbachol before staining with R-BT. For ex- of the myotubes present at the end of the experiment contained ample, similar amountsof carbachol should remain after wash- AChR clusters(see Table 2). Similar resultswere obtainedwhen ing cells treated for 2 and 6 hr, but the percentageof myotubes myotube cultureswere stainedwith R-BT after the 6 hr exposure with clustersat 6 hr is one-third that seenafter 2 hr oftreatment. to carbachol but before the 18 hr recovery period (seeTable 2). Also, d-tubocurarine at 1O-4 M causesno lossof clusters,despite Thus, all the clusters seenafter the 18 hr recovery contained the fact that it has a higher affinity for AChR than doescar- AChRs that were present before recovery began. This suggests bachol. Thus, residual carbachol doesnot compete with R-BT that the reappearanceof AChR clusters was not due to the and prevent the visualization of AChR clusters. In the experi- formation of new myotubes during the recovery period. The ments presentedbelow, I show that cluster lossis not the result possibility that massiveloss of musclecells occurred during the of irreversible loss or damagesustained by myotubes, but is, recovery period was ruled out by measuringthe number of IZ51- instead,caused by carbacholinteracting with the nicotinic AChR BT binding sites in the cultures immediately after carbachol and inducing the influx of cations, primarily Na+, into the myo- treatment and again after recovery. In two experiments, lZ51-BT tubes. binding was 34 and 4 1%greater after recovery than immediately I first addressedthe possibility that carbachol causedthe loss after the 6 hr treatment with carbachol. Together with the data of entire myotubes displaying clusters. A total of 194 cells in in Table 2, this finding rules out the possibility that the cells four myotube cultures were identified and photographed. Two left after recovery were only thosecells that resistedthe effects cultureswere incubated under control conditions, and two were of carbachol.I concludethat the carbachol-inducedloss of AChR incubatedsimilarly but in the presenceof 1O-“ M carbachol. The clusters is reversible. cultures were then stained with R-BT, and the identified cells I observed the disruptive effectsof carbachol on AChR clus- were observedand rephotographed.The results(Table 1) show ters in all the experiments (total of 59) I performed. In 12 (20%) that most of the control cellswere presentafter 6 hr of incubation of theseexperiments, however, the effect of carbachol treatment and that most of these displayed AChR clusters. Most of the wasquantitatively diminished, resultingin 40-5 5% of myotubes carbachol-treatedcells were also recovered after 6 hr, but only with clusters after 6 hr, rather than the usual 20-30%. This 4% of these had clusters. Examples are presentedin Figure 2. variability may depend on the state of development of the cul- The resultsindicate that carbachol causesAChR clusters to be tures, but I have not found a consistent effect either of culture lost without causingsignificant loss of muscle cells. age or of initial plating density. Unless otherwise stated, the Carbachol-treated cells underwent some change in shape, resultsreported hererepresent only thoseexperiments in which however (Fig. 2, A and B, white arrows). After treatment with carbachol treatment resulted in ~40% of myotubes with clus- 1O-4M carbachol, the cuspsof the myotubes appeared broader ters. The experiments that have been omitted always gave qual- and the myotube sectionsconnecting the cuspsappeared nar- itatively similarresults. Quantitatively, however, drugsthat block rower. Although these shape changesaccompany the loss of AChR cluster loss in the presenceof carbachol are generally clusters,more extensive changesin myotube shapeare induced more potent when carbachol-inducedcluster lossis diminished. by colchicine,without extensive cluster loss(Bloch, 1979, 1983), Recently, considerableattention has beengiven to the AChR suggestingthat alteration of myotube shapeis itself not sufficient clustersthat form on the dorsal or upper myotube surface,dis- to causeloss of AChR clusters.Vacuolization, observed in some tant from the tissue culture substrate.Dorsal clusters,too, are carbachol-treatedcells (Fig. 2B, black arrowhead)did not always destabilized by carbachol: In two experiments, the percent of accompanyloss of AChR clusters,as lessthan 15%of the muscle myotubes with dorsal clusters decreasedby 40 and 69%, as cells becamevacuolated during exposure to carbachol. compared to controls. The effects of carbachol on the ventral I next determined if the effect of carbachol on AChR clusters clusterswere quantitatively similar. In my cultures, dorsal clus- was reversible. After exposure to 1O-4M carbachol for 6 hr, ters are generally infrequent and more variable than are ventral cultures were washed extensively in fresh medium and incu- clusters (from 23 to 5 1% myotubes with dorsal clusters,in six The Journal of Neuroscience Carbachol-Induced Loss of Myotube ACh Receptor Clusters 693

Figure 2. The effect of carbachol on cell number and AChR clusters in identified cells. Cultures were processed as described in the legend to Table 1. At the beginning of the experiment, phase micrographs were taken of control cultures (not shown) and of cultures to which carbachol had just been added (A). Cultures were then replaced at 37°C for 6 hr. After this incubation, cultures were labeled with R-BT and fixed with parafotmaldehyde. The same fields were relocated and photographed again (& for carbachol-treated sample). The magnification was then increased, and individual control (C, D) and carbachol-treated (E, F) cells were examined and photographed under phase (C, E) and fluorescence (D, F) illumination to determine if AChR clusters were present. Panels E and F show the cell marked with the whitearrow in B. This figure shows that most cells are recovered after treatment with carbachol, but that, unlike control cells, they do not contain AChR clusters. It also illustrates some of the shape changes (compare cell indicated by the whiteuvows in A and B) and occasional vacuolization (e.g., the cell indicated by the blackarrowhead in B) that treatment with carbachol may induce. The clearer view of the nuclei in C’, compared to E, is due to the fact that the cell in C is spread more, and not to any significant change in the appearance of the myoplasm after carbachol treatment. The bar in F represents 20 pm for panels C-F, and 80 rrn for panels A and B. 694 Vol. 6, No. 3, Mar. 1986

Table 2. Reversibility of carbachol-induced loss of AChR clusters Table 3. Effect of cholinergic ligands on AChR cluster stability

Myotubes with clusters (%) Myotubes with clusters (%) Dmz After initial (concentration) No carbachol Carbachol After recovery incubation Controls 81 f 6 (21p 23 + 10 (23) No carbachoP CarbachoP No car- Car- Nicotinic agonists Experiment bachop bachoP A B A B Nicotine ( 1Oe4 M) 48 f 7 (4) -h 1 84 14 92 -b 90 -b DMPP (lo-< M)C IO,12 20,2 2 87 20 80 80 86 74 Neostigmine ( 1Oe4 M) 27 + 13(4) 28 f 12(3) 3 64 14 61 64 57 56 Decamethonium ( 1Oe5 M) 52.52 24.32 Myotube cultures were incubated with and without carbachol (10-l M) for 6 hr at Nicotinic antagonists 37°C. They were then washed extensively. Duplicate samples were withdrawn, stained with R-BT, fixed in cold ethanol, and stored until the termination of the R-BTJ 87,85 80,84 experiment. Data for these samples are given in the columns labeled “After initial d-Tubocurarine (1 O-4 M) 76 + 6 (3) 74 f lO(5) incubation.” For the samples in column A, remaining cultures were replaced in Benzoquinonium ( 10m4M) 78.74 82.76' control medium and incubated at 37°C for an additional 16-18 hr. Thereafter, they were stained with R-BT and fixed. For the samples in column B, remaining Gallamine cultures were first stained with R-BT and then replaced in control medium. After (It4 M) 73,67 48,39 the additional incubation, they were fixed without restaining. All samples were (2.5 x IO-’ M) 77 67 mounted in glycerol, and myotubes with clusters were quantitated as described in Methods. Hexamethonium a Initial incubation. (5 X It4 M) 84,76 43,so b Not determined. Nicotinic modifiers QX314 control samples) and are harder to visualize. They were, there- (2.5 x 1oF M) 82 75 fore, only examined occasionally. (5.0 X lo-4 M) 82 76,70 Meproadifen ( 1Oe5 M) 78,79 75,70 Pharmacology Fluphenazine (2 x 10m5M) 80,71 82,64 Further experiments,summarized in Table 3, showedthat the effectsof carbacholare the result of its interaction with AChR. Related compounds (1) All agonistsof the AChR, including neostigmine(Bloch and Muscarine ( 1O-4 M) 77 + 10 (3) 16 Stallcup, 1979),cause extensive cluster loss.(2) Partial agonists, Carbamyl @-methylcholine such as decamethonium (Adams and Sakmann, 1978), cause (lom4 M) 80 + 11 (3) 32 f 19(3) lessextensive cluster loss. (3) Antagonists, such as d-tubocu- Choline ( 1Om4 M) 66,71 -b rarine and benzoquinonium, alone have no effect on AChR Acetylthiocholine ( 10m4M) 26 k 12(3) 21 + 17(3) clusters,but they reduce or prevent the effect of carbachol. (4) Myotube cultures were incubated for 6 hr at 37°C with the indicated drug, alone A local anesthetic,QX3 14, which blocks the AChR ion channel or together with 10e4 M carbachol. Cultures were washed extensively to remove in its open conformation (Neher and Steinbach, 1978), blocks the ligands, stained with R-BT, fixed in cold ethanol, and mounted in glycerol. the carbachol-inducedloss of AChR clusters. (5) Meproadifen Myotubes with clusters were quantitated as described in Methods. and phenothiazines, which accelerate AChR desensitization 0 Mean +- SD, followed by the number of determinations, in parentheses. (Carp et al., 1983; Krodel et al., 1979; Maleque et al., 1983), h Not determined. alsoprevent carbachol-inducedcluster loss.Thus, reagentsthat ( DMPP, 1,l -dimethyl-4-phenylpiperazinium iodide. causeAChR channel opening causecluster loss, and reagents d Cultures were preincubated with 5 &ml R-BT for 20 min. Cultures were then that block channel opening or reduce the channel open time washed extensively and incubated for 6 hr, as for the other samples. prevent cluster loss. c Value obtained when carbachol alone gave only 49% myotubes with clusters. The effectsof different concentrationsof severalof thesedrugs arepresented in Figure 3. Carbacholcauses half-maximal cluster loss at a concentration of 3 PM (Fig. 3A). Decamethonium shows mational change in AChR that is sufficient to induce cluster a biphasic effect on AChR clusters:At 1O-5M, it causessome loss.(2) Ion flux through the receptor ion channel must occur. cluster loss, but at lo-“ M it causes no cluster loss (Fig. 3B). (3) Depolarization, which is long-lastingin carbachol-treatedrat These dual effects of decamethoniumare probably due to the myotubes(Ritchie and Fambrough, 1975a),is sufficient to cause fact (Adams and Sakmann, 1978) that, at lower concentrations, cluster loss. decamethoniuminteracts with AChR to open some channels, The results reported in Table 2 make the first possibility un- while at higher concentrations it blocks open channelsin the likely. For example, QX3 14 doesnot appear to alter the rates manner of a local anesthetic. Over the concentration range 10-8- of channel opening and closing or of receptor desensitization 1O-4 M, d-tubocurarine causesno cluster lossand blocks half (Neher and Steinbach, 1978). As QX3 14 inhibits the effectsof the effect of 1O-4M carbachol at a concentration of 7 PM (Fig. carbachol, the conformational changesassociated with these 3C’). Fluphenazine (Fig. 30) blocks carbachol-induced cluster receptor functions are apparently not sufficient to causecluster lossat the sameconcentrations at which trifluperazine, a closely loss.I therefore concentrated on the latter two possibilities. related phenothiazine, promotesreceptor desensitization(Carp To test the third possibility, I incubatedmyotubes in medium et al., 1983; Maleque et al., 1983). Qualitatively, the results containing 145mM K+. When the counterion waschloride, clus- shown in Figure 3 and Table 3 are consistent with the idea that ter losswas extensive (giving < 10% myotubeswith clusters,in the lossof AChR clustersis a nicotinic cholinergic effect. two experiments)and wasaccompanied by swellingof the myo- tubesand other morphological changes.(Additional changesin Ionic dependence the appearanceof myotubes treated with high concentrations Three distinct mechanisms could account for the effect of car- of KC1 include the presenceof more clearly defined nuclei, with bachol on AChR clusters. (1) Carbachol may induce a confor- darkenednuclear envelope, lighter nucleoplasm,and condensed The Journal of Neuroscience Carbachol-Induced Loss of Myotube ACh Receptor Clusters 695

go-

I 10-s 1 o-4

Carbachoi (Ml D go- 0 Q Figure 4. Concentration dependence of carbachol-induced 2ZNa+ in- 0 . flux into myotube cultures. Cultures were placed into a balanced salt solution (Bloch, 1983) containing radioactive Na+ at 35-37°C in the 50- presence of the indicated concentration of carbachol. After 15 min, . cultures were rapidly washed free of unbound 22Na+, and the radioac-

lo- tivity taken up per culture was determined. More details are given in -J ( Stallcup and Cohn (1976). Each point is the mean f SD ofthree samples.

10-6 10-s 10-a 10-3 The results show that over a 15 min incubation period, carbachol stim- o 10-7 ulates **Na+ uptake by myotube cultures with a half-maximal concen- Reagent(M) tration of approximately 3 ~h4. Figure 3. Concentration dependence of the effect of receptor ligands The most likely explanation of the effects of carbachol on on AChR cluster stability. Myotube cultures were incubated in medium AChR clustersis that carbachol treatment causesan influx of containing the concentration of ligand(s) indicated on the abscissa. After cations into the myotube, altering the intracellular milieu and 6 hr at 37°C cultures were washed extensively, stained with R-BT, fixed thereby destabilizing clusters.As Na+ is the predominant cation in cold ethanol, and observed under fluorescence optics. Myotubes with clusters were quantitated as described in Methods. A, Carbachol shows in culture medium that can permeatethe AChR channel(Huang a half-maximal effect on cluster stability at 2.5 PM. B, Decamethonium et al., 1978;Ritchie and Fambrough, 1975b; Takeuchi and Tak- shows a biphasic effect. C, Benzoquinonium (x) and d-tubocurarine euchi, 1960) I attempted to correlate Na+ entry into myotubes (0) alone have no effect on AChR clusters, but d-tubocurarine in the with carbachol-inducedcluster loss. I first determined the de- presence of 1O-4 M carbachol(0) inhibits cluster loss with a half-maximal pendenceof 22Na+influx on carbachol concentration, assaying effect at 7 FM. D, Trifluperazine alone (0) has no effect on cluster sta- after 15 min at 37°C as a compromisebetween conditions used bility, but in the presence of 10m4 M carbachol (0) it inhibits cluster for kinetic measurements(5-l 0 set) and thoseused to observe loss with a half-maximal effect occurring between 5 and 10 FM. cluster loss(2-6 hr). As shown in Figure 4, the concentration of carbachol that gave a half-maximal **Na+ influx was 3 MM nucleoli. These changesare not observed in myotubes treated (Fig. 3A). The similar half-maximal concentrations for cluster with carbachol or with K-methane sulfonate.) When the coun- lossand **Na+influx suggestthat the two processesare related. terionswere 4 mM Cl- and 141 mM methanesulfonate, however, Second, I found that lowering the extracellular Na+ concentra- I found no loss of AChR clusters (78 + 5% myotubes with tion reducesthe effect of carbachol. The data in Figure 5B show clusters,n = 3), and no swelling. As myotubes are depolarized that partial protection against cluster loss was obtained when to - 1 t 0.7 mV (mean + SD, n = 3 cells in a single culture, the extracellular Na+ concentration was reducedto 60 mM. At measuredwith an intracellular microelectrode)by high concen- [Na’],, lessthan 30 mM, myotubes contracted and were lost trations of K+-methanesulfonate, depolarization is not sufficient from cultures, perhaps becauseCa*+ influx through the AChR to causecluster loss. ionophore contributed more to net cation influx (e.g., Huang et I ruled out the possibility that Cl- was important for cluster al., 1978; Stallcup, 1979). Theseresults are consistentwith the loss in two separateexperiments. In medium containing 145 idea that Na+ flux into myotubes is at least in part responsible mM Na+, supplementedwith 30 mM KC1 (insufficient to cause for AChR cluster lossinduced by carbachol. cluster lossby itself), and containing - 140 mM Cl-, carbachol Two additional observations support this idea. (1) Veratri- treatment wasunable to disrupt AChR clusters(67 f 10%myo- dine, an alkaloid that specifically opensvoltage-gated Na+ chan- tubeswith clusters,n = 3). Under theseconditions, elevated K+ nels in excitable cells (Catterall and Nirenberg, 1973; Stallcup may reduceNa+ movement through the AChR ion channelby and Cohn, 1976) causesextensive AChR cluster loss.At 2.5 x competing for accessto the channel, and also by reducing the 1Om4 M, veratridine left only 2 1 + 14%(n = 3) of myotubeswith driving force for Na+ influx. In the secondexperiment, carbachol clusters. At the same time, however, it causessevere changes treatment in the presenceof 4 mM KC1and 141 mM Na-methane in cell shape.(2) Gramicidin S, an ionophore that forms specific sulfonate resulted in normal cluster loss (43% myotubes with channelsfor Na+ and K+ in cell membranes(Jain, 1972),at 10 clusters,compared to 40% in medium containing 145 mM Cl-, &ml also causessignificant AChR cluster loss(leaving 35 and in a singleexperiment). Both theseexperiments suggest that Cl- 14% myotubes with clusters in two experiments). At higher is not essentialfor cluster lossto occur in the presenceof car- concentrations, however, this reagent causesmassive contrac- bachol. tion and cell loss.Despite the difficulties involved in their use, 696 Bloch Vol. 6, No. 3, Mar. 1986

Table 4. Effect of protease inhibitors on AChR cluster loss induced by carbachol

Myotubes with Preincu- clusters (%) bation No time car- Car- Exp. 0-4 Inhibitor bachol bachol 1 1 Control 86 6 Leupeptin (25 PM) 86 26 Aprotinin (0.5 TIU/ml)’ 80 20 2 1 Control 83 19 Leupeptin (50 PM) 83 20 Aprotinin (0.5 TIU/ml) -b 12 Pepstatin (50 &ml) 72 29 3 1 Control 73 14 Leupeptin (250 PM) 70 24 4 16 Control 77 49 Leupeptin (250 FM) 66 35 Ep 475 (100 j&M) 67 42d 5 16 Control 71 23 Leupeptin (50 PM) 68 18 Ep475 (250 /L(M) 65 19 Leupeptin (50 PM) plus 59 21 Ep475 (250 FM) Ca*+(mM) Myotubes were preincubated with protease inhibitor at the desired concentration for the period of time noted. Carbachol was then added to half of the cultures to Figure 5. Effect of altering the extracellular salt concentrations on a final concentration of 1O-d M. After 6 hr of incubation at 37°C cultures were cluster loss induced by carbachol. Myotube cultures were placed into washed, stained with R-BT, fixed, and observed for AChR clusters. tissue culture medium containing the concentration of Ca2+ indicated Q The units are: TIU, trypsin inhibitory units, as defined by the manufacturer on the abscissa, and either 150 mM NaCl (closedcircles) or 60 mM NaCl (Sigma). (open squares). Isotonicity was maintained by the addition of sucrose. b Not determined. Cultures were then incubated either in the absence (A) or presence (B) c In experiment 4, carbachol had a less marked effect than in most of the other of 10m4 M carbachol for 6 hr at 37°C. After staining with R-BT and experiments reported here. Usually when this occurred, inhibitors of cluster loss fixation, AChR clusters were quantitated as described in Methods. The were more potent. In this case, however, protease inhibitors were unable to protect raw data are presented in A and B. Panel C shows the results expressed a significant number of receptor clusters from the disruptive effects of carbachol, as the percent control values, which were obtained by dividing the values despite the fact that they were used at high concentrations. found in the presence ofcarbachol (B) by the values found in its absence d This difference is within the standard range of variability for such measurements (A) under otherwise identical conditions. In agreement with other re- (see Methods). ports (Bloch, 1983; Bursztajn et al., 1984), the results show that lowering extracellular Ca2+ causes AChR cluster loss (A, solidline) and that this requires the presence of high extracellular Na+ (A, dottedline). In the clustersfrom disruption (Fig. 5B, solid line). This effect is even presence of carbachol (B and, after normalization, C), lowering extra- more apparent when normalized to account for changesin the cellular Na+ protects most of the AChR clusters from disruption (dotted control values at the same salt concentrations (Fig. SC, solid lines). This protection is extended to more myotubes with clusters when line). These experiments suggestthat [Ca2+lomay also be in- extracellular Ca*+ is lowered (solidlines). volved in the lossof AChR clusterscaused by carbachol.

both these reagents increase the permeability to Na+, and both Proteaseinhibitors cause the loss of AChR clusters.Any meansof increasingNa+ Salpeter and her colleagues(Leonard and Salpeter, 1979; Sal- influx may therefore disrupt AChR clusters. peter et al., 1982b)have reported that alterationsin postsynaptic As lowering extracellular Na+ only partially protects against morphology of musclecaused by carbachol may be due to Ca2+ clusterloss, Na+ is probably not the only cation involved, how- influx into the end-plate region and subsequentactivation of ever. As reported elsewhere(Bloch, 1983; Bursztajn et al., 1984) Ca2+-dependentneutral proteases.Carbachol-induced damage lowering [Ca2+10to concentrationsbelow 0.4 mM causescluster is reduced in musclesincubated in medium lacking Ca2+or in loss,and this lossis preventedby simultaneouslyreducing [Na’],, medium containing the proteaseinhibitor, leupeptin (Salpeter (Fig. 54). The effect of very low concentrationsof extracellular et al., 1982b). It seemedreasonable to postulate that AChR CaZ+on carbachol-inducedAChR cluster losscould therefore clusters in cultured myotubes might be disrupted by a similar not bedetermined. At [Ca2+lobetween 0.4 and 2.0 mM, however, mechanism.I therefore treated myotube cultureswith leupeptin I observedgreater protection againstthe effectsof carbachol. In or with an inhibitor specific for Ca2+-activatedthiol proteases, medium containing 60 mM Na+, decreased[Ca2+10 prevented an Ep475 (Rodemann et al., 1982), for up to 16 hr, to allow the additional lo-20% of cluster loss (Fig. 5B, dotted line). After reagentsto gain accessto the cytoplasmic compartment, then normalization for the effectsof the changesin salt concentration exposedthem to carbachol for 6 hr in the continued presence on controls, however, lower [Ca2+10at 60 mM NaCl only gave of these reagents.The results of these experiments (Table 4) additional protection againstcarbachol-induced cluster lossin showthat neither leupeptin nor Ep475 protects myotubesfrom the upper rangeof concentrationsassayed (Fig. SC, dotted line). carbachol-inducedAChR cluster loss.Aprotinin and pepstatin With [Na+loconstant at 150mM, reduced [Ca’+],,also protected (Table 4), and L-seryl+leucine (not shown),also failed to inhibit The Journal of Neuroscience Carbachol-InducedLoss of Myotube ACh Receptor Clusters 697

Table 5. Effects of other drugs and treatments on AChR cluster loss Several possibilities are highly unlikely, however. (1) Cluster induced by carbachol treatment loss is not the result of massiveloss of myotubes or of irre- versible damageto the myotubes.(2) It is probably not the result of the changesin cell shape accompanying exposure to car- Treatment or drug Myotubes with clusters (%) (concentrations) No carbachop Carbachol bachol. More extensive changesin shapeare induced by ex- posure to colchicine, for example, without significant cluster None 81 + 6 (21)“.c 23 k lO(23) loss. Furthermore, myotube-substrate contacts, at which clus- BaClz (2 mM) 19,68 46 k 6 (4) ters are often found (e.g., Bloch and Geiger, 1980; Land et al., MnCl, (2 mM) 78,68 21 -t 14(3) 1977), are not significantly altered during carbachol treatment SrCl, (2 mM) 11, 90d 25, 10 (R. J. Bloch, unpublished observations), and the organization COCI, (2 rnM) 73,62 7 2 5 (4) of membranedomains in this region of the cell is not lost as clustersare disruptedduring exposureto carbachol(D. W. Pum- MgCI, (4 mM)e 64, 86d 6, 25 plin and R. J. Bloch, unpublishedobservations). Theseobser- Ouabain (5 mM) 69,62 20,4 vations suggestthat carbachol doesnot grosslydeform the re- Colchicine ( 10m5 M) 69 k 6 (3) 22,22 gions of the myotubes at which clusters are normally found. Cytochalasin B (2 &ml) 84 f 5 (3) 31,15 (3) Cluster loss is probably not the result of the sheddingof Cycloheximide (50 &ml) 66 f 1 I (3) IO,12 clustersfrom the cell. In caseswhere this has been observed Concanavalin A ( 10 &ml) 91,83 62 + 6 (3) (e.g., Bloch, 1984, and unpublished observations),clusters are Prefixation’ 70,73 84,82 shedonto the adjacenttissue culture substrate.No suchshedding Room temperatures 76,16 84,62 wasobserved in theseexperiments. (4) Disappearanceis prob- ably not the result of carbachol remaining bound or trapped by Myotube cultures were incubated in the presence of the drugs listed, with or clusteredreceptors. Instead, the resultspresented here show that without carbachol (lo-’ M), for 6 hr at 37°C. Cultures were then washed, stained with R-BT, fixed, and analyzed for myotubes with AChR clusters. the lossof AChR clustersseen in rat myotube cultures treated a Unless otherwise indicated, the values in this column are from Bloch (1979, with carbachol is a pharmacologicaleffect mediatedby ion flux 1983). through the AChR channels. b When appropriate, results are expressed as means ? SD, followed by the number The pharmacology of cluster loss is nicotinic cholinergic. of determinations (in parentheses). Cluster loss is causedby a variety of nicotinic agonistsand 5 Values from Table 2. blocked by classicalnicotinic antagonists,including BT. It is d Values not previously reported. also inhibited by the local anesthetic,QX3 14, and by meproad- c The medium already contained 2 mM M&I,, so the final concentration was 6 ifen. As argued in Results, the effects of these drugs are not mw consistentwith the idea that carbacholcauses the lossof AChR ‘Samples were fixed for S min at 22°C in 2% (wt/vol) paraformaldehyde in buffered saline before being placed into incubation with or without carbachol. clusterssimply becauseit induces a conformational changein I: Incubations were conducted as for the other samples described, but the temperature the receptorsthat rendersthem lessable to cluster.Instead, some was kept at 22°C. effect that follows agonist-inducedconformational changesmust be responsiblefor cluster lossin the presenceof carbachol.This effect is not simply membranedepolarization, however, as K+ cluster lossin the presence of carbachol. These results suggest at high concentrations (with methane sulfonate as the counter- that carbacholdoes not causecluster lossin cultured myotubes ion) doesnot causethe lossof AChR clusters.Thus, cluster loss solely by activating Ca2+-dependentneutral proteases. probably results from the flux of particular ions through the AChR channelsopened by carbachol. Other drugs Several results suggestthat, of the cations that permeatethe To define further the possiblechanges in myotubes causedby AChR channel, Na+ is the most important. (1) Carbacholstim- carbachol, I examined the effects of several other treatments ulates *2Na+flux and cluster losswith the same half-maximal and drugs (Table 5). Only prefixation with paraformaldehyde, concentration. (2) Two other reagentsthat stimulateNa+ influx, incubation at room temperature, or incubation in the presence veratridine and gramicidin, also causeAChR cluster loss. (3) of concanavalin A protected against cluster loss, perhaps by Reducing extracellular Na+ concentrationsprevents much clus- reducingthe mobility of AChR in the myotube membrane.Ba2+, ter loss.This interpretation is also consistentwith the observed but no other divalent cation, gavepartial protection. Colchicine, effect of electrical activity on AChR clusters.For example, Ax- which partially protects againstAChR cluster lossin Caz+-free elrod et al. (1978) showedthat repeated electrical stimulation medium (Bloch, 1983), had no effect. Ouabain, which should of myotube cultures causedthe AChR clustersto be lost. Such potentiate the increasein intracellular Na+ initiated by car- stimulation would tend to increaseintracellular Na+. Several bachol, increasedboth cluster lossand lossof myotubes due to investigators have further reported that TTX, which should contraction. Other drugsthat had no effect on the lossof clusters block spontaneousopening of the voltage-activated Na+channel inducedby carbacholinclude 3’,5’-CAMP, 3’,5’-cGMP, and their and thereby prevent an increasein intracellular Na+ levels due dibutyryl derivatives, all assayedat 1 mM in the presenceor to suchopenings, promotes the formation of AChR clusters(e.g., absenceof theophylline (1 mM), theophylline alone (1 mM), Harris, 1981; Prives et al., 1976;Ziskind-Conhaim and Bennett, caffeine(2 mM), dimethylsulfoxide (lo/o),and ethanol (1%). Oth- 1982). Treatments that increase[Na+li are therefore likely to er neurotransmitteragonists and antagonistsalso failed to alter disrupt AChR clusters,and treatmentsthat block suchincreases AChR cluster stability. Reagentstested include histamine, gly- are likely to promote cluster formation. tine, glutamate,y-aminobutyric acid, epinephrine, norepineph- One difficulty with this interpretation is that the concentra- rine, and serotonin, all at 10d4M. tions of nicotinic drugs neededto affect AChR clustersdo not consistently agree with published values of their affinities for Discussion AChR on the surface of mammalian cells (e.g., Patrick et al., 1977; Sineand Taylor, 1979, 1981). Thesedifferences are prob- Nicotinic cholinergic eflect ably the consequenceof studying long-term effects, involving Exposureof cultured rat myotubes to carbachol causesthe dis- processesother than AChR channel opening (e.g., a gradual appearanceof AChR clustersfrom the myotube membrane.The reduction in the driving force for Na+ influx or a changein the mechanismof this disappearance,or loss, is still not known. activity of the Na+-K+ pump). A similar half-maximal concen- 698 Bloch Vol. 6, No. 3, Mar. 1986

tration for carbachol has been reported by Lindstrom et al. synaptic Ca2+concentrations can activate neutral proteases,and (1980) who studied 22Na+flux into small vesicles,stimulated that this causesdisruption ofpostsynaptic morphology. To learn by carbachol interacting with AChR, at times much later than if similar enzymes participate in cluster loss in rat myotubes those required to observe true initial velocities. treated with carbachol, I treated myotube cultureswith specific A second possible difficulty with this interpretation is the protease inhibitors. Such treatments gave little protection of observation by Ziskind and Dennis (1978) and others (Morris clusters(Table 4). Ca2+-activatedneutral proteasesare therefore et al., 1983; Trautmann, 1982) that d-tubocurarine can act as probably not involved. a nicotinic agonistin embryonic rat muscle.Ziskind and Dennis Peng(1984) has suggestedthat calmodulin might be involved (1978)report, however, that bath application of d-tubocurarine in regulation of AChR clustering. This possibility is difficult to to embryonic muscle depolarizes embryonic muscle fibers to assess.All organic inhibitors of calmodulin, including fluphen- -50 mV. In contrast, carbachol causeslong-lasting depolari- azine (Landry et al., 1981; Weisset al., 1980),exert their effects zation of cultured rat myotubes to 0 to - 10 mV (Ritchie and on calmodulin at micromolar concentrations. At these same Fambrough, 1975a).Thus, d-tubocurarine probably fails to dis- concentrations, the phenothiazines promote AChR desensiti- rupt clustersbecause it is not a sufficiently potent receptor ag- zation (Carp et al., 1983; Maleque et al., 1983), a processthat onist. alone should protect against cluster loss causedby carbachol. In the same concentration range, these and other calmodulin Role of Ca2+ inhibitors can also act as local anesthetics(e.g., Creeseet al., Although Na+ flux through the AChR channel appearsto be 1978; Seeman,1972). It is therefore difficult to distinguishthe important for cluster lossto occur in the presenceof carbachol, effectsof thesedrugs on calmodulin from their effectson AChR it may not be sufficient. Some protection againstcluster lossis and on general membranestructure. afforded by reducing extracellular Ca*+concentrations two- to In summary, it is not yet clear how or where Ca2+participates fourfold, and nearly completeprotection is afforded by reducing in the disruption of AChR clustersinduced by carbachol, nor both Na+ and Ca2+.In addition, Ba2+ partially blocks cluster is the identity of the macromoleculesthat mediate the action loss(Table 5) without interfering with carbachol-stimulated22Na+ of Caz+apparent. influx (Bloch, unpublishedobservations). BaZ+ is more likely to act at divalent than at monovalent cation binding sites. I ten- tatively conclude, therefore, that both Na+ and Ca2+promote Local effectsof carbachol AChR cluster lossin the presenceof carbachol. Although carbachol may interact equally well with AChR any- How doesthe movement of Na+ through the AChR ion chan- where in the musclemembrane, it may promote cluster lossby nel, in the presenceof extracellular Ca2+,destabilize AChR clus- interacting with the receptorswithin clusters.About 10% of the ters?Answering this question requiresthat we know where Ca2+ total number of AChR in culturesof rat myotubesare in clusters, acts. In principle, its site of action could be intracellular, extra- where they are presentat densities1 O-fold higher than receptors cellular, or within the plasmamembrane. If Ca2+acts intracel- elsewhereon the cell surface(Axelrod et al., 1976; Land et al., lularly, its intracellular concentrations may be elevated via a 1977; Salpeteret al., 1982a). When theseclustered AChR bind Na+-for-Ca*+exchange mechanism (e.g., Blaustein and Nelson, carbachol,the local ionic changesthat result may be greaterthan 1981) driven by high [Na+],. Other investigators and I have those occurring away from clusters. This may help to explain arguedthat depletion of intracellular Ca*+causes disruption of some of the differences between myotubes treated with car- the AChR clustersof cultured rat myotubes(Bloch, 1983;Bursz- bachol and those treated with veratridine or gramicidin. The tajn et al., 1984). If a rise in intracellular Ca2+is an important former changeshape slightly (e.g., Fig. 1), but the latter tend to factor in carbachol-inducedcluster loss,then, to account for all swell and contract. Both veratridine and gramicidin would prob- my results, receptor clustering would have to show a “bell- ably promote Na+ flux more or lessuniformly aroundthe muscle shaped”or biphasicdependence on intracellular Ca2+.This would membrane.They may causerelatively uniform increasesin ionic be consistentwith the data in Figure 5C(solid line). A disruptive concentration throughout the myoplasm and induce shape effect on clustersof increasedintracellular Ca2+would also be changesor contraction at the same drug concentrations that consistentwith observations(Bloch, unpublishedobservations) causecluster loss. Because its effectswould be enhancedin areas that the ionophore, A23 187 at concentrations that causecon- of high AChR density, carbachol might alter cluster stability traction and lossof some myotubes, causescluster lossin most before it grosslyaffected cell shape. remainingcells. However, in two experiments usingcarbachol, The potentially disruptive effect of carbacholon AChR clus- I have failed to observeincreased 45Ca2+ influx into myotubes ters suggeststhat only low levels of neurotransmissionmay be (unpublishedobservations). It is therefore possible that CaZ+ tolerated at early stagesof development of the neuromuscular influx doesnot increaseduring carbachol treatment. junction. This isconsistent with severalobservations. The initial It is difficult, nevertheless,to account for the effect of Ca*+ formation of AChR aggregatesat the developing postsynaptic by postulating that it acts either extracellularly or at the level membranedoes not require functional neurotransmission(An- of the plasmamembrane. Basedon their observation that high derson and Cohen, 1977; Anderson et al., 1977; Dennis et al., extracellular Ca2+induces cluster growth, Bursztajn et al. (1984) 1981). Electrical or synaptic activity more intensethan that seen suggestedthat Ca2+might promote interaction of the muscle at developing synapses(Dennis et al., 1981) seemsto prevent membrane with “extracellular elements,” thereby stabilizing or reverse the formation of postsynaptic receptor aggregates. AChR clusters. Several authors have also suggestedthat in- For example, electrical stimulation suppressesreceptor accu- creasedlocal concentrations of Ca2+might promote AChR de- mulation at newly forming ectopic synapsesin denervated adult sensitization(Magazanik and Vyskocil, 1970; Manthey, 1966; muscle(Lsmo and Slater, 1978). Also, as with cultured myo- Nastukand Parsons,1970). The observation that lowering, rath- tubes, exposureof the intact, embryonic rat muscleto carbachol er than raising, extracellular Ca2+protects againstcluster lossis causesthe disappearanceof postsynapticAChR aggregates(Bloch inconsistentwith the possibility that changesin theseinterac- and Steinbach, 1981). During development, as the frequency tions account for the loss of AChR clusters induced by car- and intensity of transmission at the neuromuscularjunction bachol. increase(see Diamond and Miledi, 1962; Kidokoro et al., 1980; Other investigators have suggestedspecific moleculesthat Kullberg et al., 1977), the postsynaptic receptor aggregatesbe- might mediate the affect of Ca*+on the stability of AChR-rich come more resistant to disruption by electrical stimulation or membrane. Salpeterand her colleagues(Leonard and Salpeter, cholinergic agonists(Bloch and Steinbach, 1981; Lomo and Sla- 1979; Salpeter et al., 1982b) have shown that increasedpost- ter, 1978). The Journal of Neuroscience Carbachol-induced Loss of Myotube ACh Receptor Clusters 699

Conclusion ciated with activation of action potential ionophores of cultured neu- Increasedinflux of Na+, induced by carbachol or by other treat- roblastoma and muscle cells. Proc. Natl. Acad. Sci. USA 70: 3759- 3763. ments,probably destabilizesAChR clusters.The mechanismof Creese, I., D. R. Burt, and S. H. Snyder (1978) Biochemical actions destabilization is not yet understood, but two possibilitiesare of neuroleptic drugs: Focus on the dopamine receptor. In Handbook worth investigating further. (1) Carbachol could indwcethe in- ofPsychopharmacology, Vol. 10, L. L. Iversen, S. D. Iversen, and S. ternalization and degradation (down-regulation) of AChR in H. Snyder, eds., pp. 39-42, Plenum, New York. primary rat myotubes, as has beenreported for chick myotubes Dennis, M. J., L. Ziskind-Conhaim, and A. J. Harris (1981) Devel- and a mouse muscle cell line by others (Gardner and Fam- opmentof neuromuscularjunctions in rat embryos.Dev. Biol. 81: brough, 1979; Noble et al., 1978). Work now in progresshas 266-279. shown that exposure of rat myotube cultures to carbachol re- Diamond, J., and R. Miledi (1962) A study of foetal and new-born ducesthe overall number of lZSI-BTbinding sitesby about 30%, rat muscle fibers. J. Physiol. (Lond.) 162: 393-408. Fambrough, D. M. (1979) Control ofacetylcholine receptors in skeletal but neither the time course nor the pharmacology of this de- muscle. Physiol. Rev. 59: 165-227. creasecorrelates with that observed for the lossof AChR clusters Gardner, J. M., and D. M. Fambrough (1979) (Bloch, unpublishedobservations). The possibility remainsthat degradation measured by density labeling: Effects of cholinergic li- carbacholcauses cluster lossby “down-regulating” the clustered gandsand evidence against recycling. Cell 16: 661-674. AChRs, which represent only about 10% of the total lZSI-BT Harris, A. J. (198 1) Embryonic growth and innervation of rat skeletal binding siteson the cell surface.(2) AChR may be stabilized in muscles. III. Neural regulation ofjunctional and extrajunctional ace- clustersby binding to intracellular cytoskeletal structures (see tylcholine receptor clusters. Phil. Trans. R. Sot. London [Biol.] 293: Steinbachand Bloch, in press,for a review). IncreasedNa+ influx 287-314. could reducethis binding or alter the cytoskeletal organization Huang, L.-Y. M., W. A. Catterall, and G. Ehrenstein (1978) Selectivity of cations and nonelectrolytes for acetylcholine-activated channels in in the vicinity of clusters. Recent progressin isolating AChR cultured muscle cells. J. Gen. Physiol. 71: 397-410. clustersfrom rat myotube cultures by mild detergentextraction Jain, M. K. (1972) The Bimolecular Lipid Membrane, p. 180, Van (Bloch, 1984)should permit theseand other possibilitiesto be NostrandReinhold, New York. investigated in the near future. Kidokoro, Y., M. J. Anderson, and R. Gruener (1980) Changes in synaptic potential properties during acetylcholine receptor accumu- lation and neurospecific interactions in Xenopus nerve-muscle cell References culture. Dev. Biol. 78: 464-483. Adams, P. R., and B. Sakmann (1978) Decamethonium both opens Krodel, E. K., R. A. Beckman, and J. B. Cohen (1979) Identification and blocks endplate channels. Proc. Natl. Acad. Sci. USA 75: 2994- of local anesthetic binding site in nicotinic postsynaptic membranes 2998. isolated from Torpedo marmoruta electric tissue. Mol. Pharmacol. Anderson, M. J., and M. W. Cohen (1977) Nerve-induced and spon- 15: 294-312. taneous redistribution of acetylcholine receptors on cultured muscle Kullberg, R. W., T. L. Lentz, and M. W. Cohen (1977) Development cells. J. Physiol. (Lond.) 268: 757-773. of the myotomal neuromuscular junction in Xenopus luevis: An elec- Anderson, M. J., M. W. Cohen, and E. Zorychta (1977) Effects of trophysiological and line-structural study. Dev. Biol. 60: 101-l 29. innervation on the distribution of acetylcholine receptors on cultured Land, B. R., T. R. Podleski, E. E. Salpeter, and M. M. Salpeter (1977) muscle cells. J. Physiol. (Lond.) 268: 73 l-756. Acetylcholine receptor distribution on myotubes in culture correlated Axelrod, D., P. Ravdin, D. E. Koppel, J. Schlessinger, W. W. Webb, E. to acetylcholine sensitivity. J. Physiol. (Lond.) 269: 155-l 76. L. Elson, and T. R. Podleski (1976) Lateral motion of fluorescently Landry, Y., M. Amellal, and M. Ruckstuhl (1981) Can calmodulin labeled acetylcholine receptors in membranes of developing muscle inhibitors be used to probe calmodulin effects?Biochem. Pharmacol. fibres. Proc. Natl. Acad. Sci. USA 73: 4594-4598. 30: 203 l-2032. Axelrod, D., P. M. Ravdin, and T. R. Podleski (1978) Control of Leonard, J. P, and M. M. Salpeter (1979) Agonist-induced myopathy acetylcholine receptor mobility and distribution in cultured muscle at the neuromuscular junction is mediated by calcium. J. Cell Biol. membranes. A fluorescence study. Biochim. Biophys. Acta 511: 23- 82: 811-819. 38. Lindstrom, J., R. Anholt, B. Einarson, A. Engle, M. Osame, and M. Blaustein, M. P., and M. T. Nelson (198 1) Sodium-calcium exchange: Montal (1980) Purification of acetylcholine receptors, reconstitution Its role in the regulation of cell calcium. In Calcium Transport Across into lipid vesicles, and study of agonist-induced cation channel reg- Biological Membranes, E. Carafoli, ed., pp. 2 17-236, Academic, New ulation. J. Biol. Chem. 255: 8340-8350. York. Lomo, T., and C. R. Slater (1978) Control of acetylcholine sensitivity Bloch, R. J. (1979) Dispersal and reformation ofacetylcholine receptor and synapse formation by muscle activity. J. Physiol. (Lond.) 275: clusters of cultured rat myotubes treated with inhibitors of energy 39 l-402. metabolism. J. Cell Biol. 82: 626-643. Magazanik, L. G., and F. Vyskocil (1970) Dependence ofacetylcholine Bloch, R. J. (1983) Acetylcholine receptor clustering in rat myotubes: desensitization on the membrane potential of frog muscle fibre and Requirement for Ca*+ and effects of drugs which depolymerize mi- on the ionic changes in the medium. J. Physiol. (Lond.) 210: 507- crotubules. J. Neurosci. 3: 2670-2680. 518. Bloch, R. J. (1984) Isolation of acetylcholine receptor clusters from Maleque, M. A., C. Souccar, J. B. Cohen, and E. X. Albuquerque (1983) cultured rat myotubes using saponin. J. Cell Biol. 99: 1084-1093. Meproadifen reaction with the ionic channel of the acetylcholine re- Bloch, R. J., and B. Geiger (1980) The localization of acetylcholine ceptor: Potentiation of agonist-induced desensitization at the frog receptor clusters in areas of cell-substrate contact in cultures of rat neuromuscular junction. Mol. Pharmacol. 22: 636-647. myotubes. Cell 21: 25-35. Manthey, A. A. (1966) The effect of calcium on the desensitization of Bloch, R. J., and W. B. Stallcup (1979) Agonist action of neostigmine membrane receptors at the neuromuscular junction. J. Gen. Physiol. on acetylcholine receptors of cultured mammalian muscle. Brain Res. 49: 963-976. 172: 378-381. Morris, C. E., B. S. Wong,M. B. Jackson,and H. Lecar (1983) Single- Bloch, R. J., and J. H. Steinbach (198 1) Reversible loss ofacetylcholine channel currents activated by curare in cultured embryonic rat muscle. receptor clusters at the developing rat neuromuscular junction. Dev. J. Neurosci. 3: 2525-253 1. Biol. 81: 386-391. Nastuk, W. L., and R. L. Parsons (1970) Factors in the inactivation Bursztajn, S., J. L. McManaman, and S. H. Appel (1984) Organization of postjunctional membrane receptors of frog skeletal muscle. J. Gen. of acetylcholine receptor clusters in cultured rat myotubes is calcium Physiol. 56: 2 18-249. dependent. J. Cell Biol. 98: 507-517. Neher, E., and J. H. Steinbach(1978) Localanesthetics transiently Carp, J. S., R. S. Aronstam, B. Witkop, and E. X. Albuquerque (1983) block currents through single acetylcholine receptor channels. J. Phys- Electrophysiological and biochemical studies on enhancement of de- iol. (Lond.)277: 153-176. sensitization by phenothiazine neuroleptics. Proc. Natl. Acad. Sci. Noble, M. D., T. H. Brown, and J. H. Peacock (1978) Regulation of USA 80: 310-314. acetylcholine receptor levels by a cholinergic agonist in mouse muscle Catterall, W. A., and M. W. Nirenberg (1973) Sodium uptake asso- cell cultures. Proc. Natl. Acad. Sci. USA 75: 3488-3492. 700 Bloch Vol. 6, No. 3, Mar. 1986

Patrick, J., J. McMillan, H. Wolfson, and J. C. O’Brien (1977) Ace- Sine, S., and P. Taylor (1979) Functional consequences of agonist- tylcholine receptor metabolism in a nonfusing muscle cell line. J. Biol. mediated state transitions in the cholinergic receptor. Studies in cul- Chem. 252: 2143-2153. tured muscle cells. J. Biol. Chem. 254: 33 15-3325. Peng, H. B. (1984) Participation of calcium and calmodulin in the Sine, S. M., and P. Taylor (198 1) Relationship between reversible formation of acetylcholine receptor clusters. J. Cell Biol. 98: 550- antagonist occupancy and the functional capacity of the acetylcholine 557. receptor. J. Biol. Chem. 256: 6692-6699. Prives, J., I. Silman, and A. Amsterdam (1976) Appearance and dis- Stallcup, W. B. (1979) Sodium and calcium fluxes in a clonal nerve annearance of acetylcholine receptor during differentiation of chick cell line. J. Physiol. (Lond.) 286: 525-540. skeletal muscle in vitro. Cell 7: 543-550. - Stallcun. W. B.. and M. Cohn (1976) Electrical properties of a clonal Ravdin, P., and D. Axelrod (1977) Fluorescent tetramethylrhodamine cell l%e as determined by measurement of ion ‘fluxes. Exp. Cell Res. derivatives of cY-bungarotoxin: Preparation, separation and charac- 98: 277-284. terization. Anal. Biochem. 80: 585-592; 83: 336 (Erratum). Steinbach, J. H., and R. J. Bloch (in press) Control of acetylcholine Ritchie, A. K., and D. M. Fambrough (1975a) Ionic properties of the receptor distribution in vertebrate skeletal muscle. In Receptorsin acetvlcholine recentor in clutured rat myotubes. J. Gen. Physiol. 65: CellRecognition and Differentiation, G. B. Price and R. Gorczynski, 7511767. - eds., Academic, New York. Ritchie, A. K., and D. M. Fambrough (1975b) Electrophysiological Takeuchi, A., and N. Takeuchi (1960) On the permeability of end- properties of the membrane and acetylcholine receptor in developing plate membrane during the action of transmitter. J. Physiol. (Lond.) rat and chick myotubes. J. Gen. Physiol. 66: 327-355. 154: 52-67. Rodemann, H. P., L. Waxman, and A. L. Goldberg (1982) The stim- Trautmann, A. (1982) Curare can open and block ionic channels as- ulation of protein degradation in muscle by Cal+ is mediated by sociated with choline& receptors. Nature 298: 272-275. prostanlandin E, and does not require the calcium-activated protease. Weiss, B., W. Prozialeck, M. Cimino, M. S. Bamette, and T. L. Wallace j. Bio< Chem. 2.57: 8716-8723.- (1980) Pharmacological regulation of calmodulin. Ann. NY Acad. Saloeter. M. M.. J. P. Leonard. and H. Kasprzak (1982a) Agonist- Sci. 356: 3 19-345. induced postsynaptic myopathy. Neurosci.Comment. 1:‘73-83. Ziskind, L., and M. J. Dennis (1978) Depolarizing effect of curare on Salpeter, M. M., S. Spanton, K. Holley, and T. R. Podleski (1982b) embryonic rat muscles. Nature 276: 622-623. Brain extract causes acetylcholine receptor redistribution which mim- Ziskind-Conhaim, L., and J. I. Bennett (1982) The effects of electrical ics some early events at developing neuromuscular junctions. J. Cell inactivity and denervation on the distribution of acetylcholine recep- Biol. 93: 417-425. tors in developing rat muscle. Dev. Biol. 90: 185-197. Seeman, P. (1972) The membrane actions of anesthetics and tran- quilizers. Pharmacol. Rev. 24: 583-655.