Histrionicotoxin Enhances Agonist-Induced Desensitization of Acetylcholine Receptor

Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5754-5758, December 1977 Neurobiology Histrionicotoxin enhances agonist-induced desensitization of acetylcholine receptor (22Na+ uptake/'251-labeled a-bungarotoxin binding) WOLFGANG BURGERMEISTER*, WILLIAM A. CATTERALLtt, AND BERNHARD WITKOP* * Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland, 20014; and t Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, NMaryland, 20014 Contributed by Bernhard Witkop September 6, 1977

ABSTRACT Dihydroisohistrionicotoxin inhibits acetyl- of Health, who isolated it from the skin of Dendrobates his- choline receptor-dependent 22Na+ uptake of cultured chick trionicus (1). a-Bungarotoxin was purified from Iyophilized muscle cells with a KI of 0.2 1AM. The inhibition is noncompetitive with respect to agonists. The toxin enhances desensitization venom of Bungarus multicinctus (Miami Serpentarium) and of the receptor by agonists which is accompanied by a 10-fold radio-iodinated as described by Vogel et al. (9). Chemicals were increase in receptor affinity for agonists. Dihydroisohistrioni- obtained from the following sources: carbamylcholine chloride, cotoxin increases the affinity of the desensitized form of the acetylcholine chloride, d-tubocurarine chloride, ouabain, and receptor for agonists but not antagonists. The results suggest that D-arabinofuranosylcytosine from Sigma; decamethonium dihydroisohistrionicotoxin inhibits the acetylcholine receptor bromide and gallamine triethiodide from K & K Laboratories; by causing an increase in the affinity of the desensitized form of the receptor for agonists and thereby stabilizing the de- nicotine dihydrochloride from Baker; F3H]leucine from New sensitized state. England Nuclear; and calf skin collagen from Calbiochem. Chick embryo extract was prepared as described in ref. 10. The pharmacological properties of histrionicotoxins (HTX), Other chemicals used for tissue culture were obtained from the alkaloids isolated from skin extracts of the tropical South same sources as described in ref. 8. American frog, Dendrobates histrionicus (1), have been studied Muscle Culture. Suspensions of single muscle cells from thigh by using electrophysiological and biochemical approaches. In muscle of 11-day-old chick embryos were prepared essentially mammalian and amphibian nerve-muscle preparations, HTX, as described by Fischbach (11). Cells were seeded at a density as well as its analogs dihydroisohistrionicotoxin (H2-HTX) and of 40,000/cm2 in collagen-coated multiwells (Costar 1.6 cm dodecahydrohistrionicotoxin (H12-HTX), act as reversible diameter) and grown as described (8). Cultures were used after blockers of neuromuscular transmission (2-4). They also block 6-9 days in vitro. Two days before use the cultures were treated noncompetitively the depolarization produced by carbamyl- with feeding medium supplemented with 10 pM D-arabino choline on the isolated electroplax of Electrophorus electricus furanosylcytosine to reduce the fibroblast population (11) and (3, 5). Binding of [3Mlacetylcholine (5) and a-i3Hlbungarotoxin with 0.2 ACi of [l3Hlleucine per ml to allow use of 3H cpm as a (6) to the nicotinic acetylcholine receptor is inhibited by HTX measure of protein recovery in experiments. and its analogs only at concentrations 10-fold greater than those Measurement of 22Na+ Uptake. Rates of uptake of 22Na+ required for inhibition of depolarization. At concentrations that into cells in monolayer cultures were measured at 360 as de- inhibit depolarization, HTX causes a small enhancement of the scribed (8, 12). The cultures were preincubated at 36° with binding of 13Hjacetylcholine (5). Binding studies with [f131- buffer 1 150 mM N-2-hydroxyethylpiperazine-N'-2-ethane- H12-HTX revealed that the toxin binds with a high affinity to sulfonic acid (Hepes)/13 mM NaCl/122 mM KCI/1.8 mM a protein fraction from Torpedo ocellata electric organ that CaC12/0.8 mM MgSO4/5.5 mM glucose, adjusted to pH 7.4 could be separated from the acetylcholine binding protein (7). with Tris] containing the effectors noted in the figure legends From the results mentioned so far it was postulated that HTX and 5 mM ouabain for inhibition of the active extrusion of Na+ acts by binding to the ion conductance modulator associated catalyzed by Na+- and K+-activated ATPase. These conditions with the acetylcholine receptor (2, 4, 7) or as an allosteric ligand for preincubation were chosen so that the ionic composition of of the acetylcholine receptor itself (3, 5). the external medium was identical to that inside the cells. Under As shown by Catterall (8), cultured embryonic chick muscle these conditions, preincubation with agonists causes no change cells provide a suitable system for studying ligand binding as in the ionic concentration gradients or the membrane poten- well as Na+ transport activity of the nicotinic acetylcholine tial. receptor. The receptor-dependent 22Na+ uptake of the cells is Uptake was initiated by incubating the cultures with buffer desensitized during exposure to carbamylcholine (8). In the 2 (50 mM Hepes/130 mM NaCl/5.4 mM KCl/1.8 mM present investigation we have used cultured muscle cells to CaC12/0.8 mM MgSO4/5.5 mM glucose/1.0 mM NaH2PO4, study effects of H2-HTX on the activation and desensitization adjusted to pH 7.4 with NaOH) containing 5 yCi of 22NaCl per of the acetylcholine receptor and on the binding of cholinergic ml, 0, 1, or 10 mM carbamylcholine, 5 mM ouabain, and the ligands to the receptor. effectors noted in the figure legends. After 30 sec at 360, uptake was terminated by removing the radioactive uptake buffer and MATERIALS AND METHODS Dihydroisohistrionicotoxin was kindly provided by John W. Abbreviations: HTX, histrionicotoxin; H2-HTX, dihydroisohistrioni- Daly, Laboratory of Chemistry, NIAMDD, National Institutes cotoxin; H12-HTX, dodecahydrohistrionicotoxin; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; buffer 1, 50 mM The costs of publication of this article were defrayed in part by the Hepes/13 mM NaCI/122 mM KCl/1.8 mM CaCl2/0.8 mM MgSO4/ payment of page charges. This article must therefore be hereby marked 5.5 mM glucose, adjusted to pH 7.4 with Tris. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate f Present address: Department of Pharmacology, School of Medicine, this fact. University of Washington, Seattle, WA 98105 5754 Neurobiology: Burgermeister et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5755

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50- - Log molrity carhamylcholine Fl(;. 2. Desensitization of receptor-dependent Na+ transport 9 8 7 6 5 activity by carbamylcholine in the absence and presence of H2-HTX. ---Log molarity H2-HTX Muscle cells were preincubated for 30 min with the indicated con- Fi(u. 1. Inhibition of receptor-dependent Na+ transport by centrations of carbamylcholine (0), carbamvlcholine plus 0.05 pM H2,-HTX. Muscle cells were preincubated for 30 min with the indi- H,-HTX (A), or carbamylcholine plus 0.1 pM H9-HTX (0). 22Na+ cated concentrations of H ,-HTX. 22Na+ uptake was then measuired uptake was then measured for S30 sec in the presence of 1 mM car- for 30 sec in the presence of 1 mM (0) or 10 mM ( A) carbamylcholine bamylcholi ne. and the indicated concentrations of H9,-HTX. This indicates that H2-HTX is a noncompetitive inhibitor of the receptor stimulation by carbamvlcholine. by washing three times at 360 with buffer 3 (164 mM NaCI/5.4 As was shown by Catterall (8), the receptor-dependent Na+ mM KCI/1.8 mM CaCJ2/0.8 mM MgSO4/5.0 Hepes, adjusted when the cultures mM transport activity desensitizes rapidly are to pH 7.4 with NaOH) containing 1 d-tubocurarine to exposed to carbamylcholine. Fig. 2 shows how the degree of inhibit receptor-activated ion movements. Cell monolayers desensitization depends on the concentration of carbamyl- were then suspended in 0.4 M NaOH and radioactivity was of 30 in scintillation counter. rates in nmol of choline that is present during a preincubation period min. measured a Uptake Half-maximal desensitization is caused by 40 MiM carbamyl- 22Na/min per mg of cell protein were calculated from the choline. By contrast, half-maximal activation of receptor- measurements of 22Na+ taken up and determinations of cell dependent Na+ transport requires approximately 400 jM protein by a modification of the method of Lowry et al. (13). carbamylcholine both under these experimental conditions and Values were corrected for variable protein recovery during the those described previously (8). Therefore, the apparent affinity assay and washing procedures on the basis of the [3H]leucine for desensitization of the receptor by carbamylcholine is 10-fold radioactivity recovered in each sample. Carbamylcholine- greater than the apparent affinity for activation of the receptor dependent 22Na+ uptake was determined as the difference of of by carbamylcholine. uptake measured in the presence and absence carbamyl- In the presence of 0.05 pMNi and 0.1 jIM H2-HTX, half-max- choline, and has been plotted in the figures. imal desensitization was observed at 14 pM and 10 pM car- Binding of 1125IJMono- and [1251J-Diiodo-a-bungarotoxin. bamylcholine, respectively (Fig. 2). Thus, H2-HTX enhances Binding experiments were carried out at 36° as described (8), the carbamylcholine-induced desensitization and causes a shift with buffer 1 supplemented with 1 mg of bovine serum albu- of the desensitization against concentration profile to lower min per ml. Excess toxin was eluted with three (if 3 nM bun- concentrations. When the cells were preincubated for 30 min garotoxin was used) or five (if >3 nM bungarotoxin was used) with 20 ,M carbamvlcholine plus increasing concentrations of I-min washes at 360 with buffer 4 (163 mM choline chloride/ H2-HTX and then assayed in the presence of H2-HTX, the in- 1.8 mM CaCI2/0.8 mM MgSO4/5 mM Hepes, adjusted to pH hibition in comparison to the H.2-HTX concentration profile 7.4 with Tris). Cells were suspended in 0.4 M NaOH, and ra- (Fig. 1) was also shifted to lower concentrations, indicative of dioactivity was measured in a well counter. Nonspecific bun- a 5-fold decrease in the K1 of H2-HTX (data not shown). garotoxin binding was determined in the presence of I mM By preincubating the cultures with carbamylcholine for short d-tubocurarine. It was less than 10% in all experiments. times, the rate of desensitization could be measured (Fig. 3). Eighty-six percent and 70% desensitization developed during RESULTS a 20-min exposure to 100 ,uM of carbamylcholine and 20 pM Effect of H2-HTX on acetylcholine receptor-dependent carbamylcholine plus 0.05 pM H2-HTX, respectively, while 20 Na+ transport pM carbamylcholine alone did not cause desensitization (Fig. Muscle cell cultures were preincubated with the toxin for at 3A). Therefore, 0.05 ,M H2-HTX enhances both the rate and least 10 min. 22Na uptake was then measured during a 30-sec extent of desensitization. When Ca2+-deficient buffer 1 was incubation with carbamylcholine, 22Na, and H2-HTX. Under used during the preincubation, the desensitization produced these conditions the acetylcholine receptor-dependent Na+ by 100 pM carbamylcholine was only 44%, while 20 pM car- transport could be completely inhibited by H2-HTX (Fig. 1). bamylcholine together with 0.05 ,uM H2-HTX produced only When 1 jiM H2-HTX was present only during the 30-sec assay 20% desensitization. Thus, as has been shown for the neuro- period, its inhibitory effect was nevertheless completely ex- muscular junction and for electric organ (14, 15), Ca2+ enhances pressed (data not shown). This indicates that the binding of desensitization. Comparison of Fig. 3 A and B also indicates that H2-HTX is a very rapid process. The apparent inhibition con- Ca2+ enhances the effect of H2-HTX on desensitization. stant of H2-HTX is a very rapid process. The apparent inhibition Effect of H2-HTX on acetylcholine receptor affinity constant of H2-HTX was almost independent of the concen- for agonists and antagonists tration of carbamylcholine used for stimulation of the acetyl- 1251-Labeled (Y-bungarotoxin has been introduced as a ra- choline receptor. With 1 mM carbamvlcholine, a K1 of 0.19 nM dioactive ligand for measuring binding of cholinergic agonists was observed; wvith 10 m\M carbamyicholine, a KI of 0.10 pM. and antagonists to the acetylcholine receptor of muscle cells (9). 5756 Neurobiology: Burgermeister et al. Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Binding of acetylcholine receptor ligands in the absence and presence of H2-HTX Apparent KD, gM Ligand No H2-HTX 1 AM H2-HTX Ratio Carbamylcholine 14 1.4 10 Acetylcholine 0.63 0.042 15 Nicotine 2.5 0.1 25 Decamethonium 0.17 0.02 8.5 d-Tubocurarine 0.8 0.8 1 Gallamine 1.3 0.9 1.4 0 10 20 0 10 20 Apparent KD values were determined by inhibition of labeled Min preincubation bungarotoxin binding to cells, as described in the legend of Fig. 4. FIG. 3. Time course of desensitization of receptor-dependent Na+ transport activity. (A) Muscle cells were preincubated, first for 10 min Na+ transport (Fig. 1) have no effect on bungarotoxin binding, with buffer 1 (0, A) or buffer 1 containing 0.05 ,uM H2-HTX (o), then for the indicated times with 20 ,uM (0) or 100 ,M carbamylcholine suggesting that the direct effect of H2-HTX on bungarotoxin (A) or 20 ,M carbamylcholine plus 0.05 ,M H2-HTX (0). 22Na+ binding is unrelated to the mechanism of action of H2-HTX. uptake was then assayed in the presence of 1 mM carbamylcholine Low concentrations of H2-HTX have a large effect on the in- (O A) or 1 mM carbamylcholine plus 0.05 gM H2-HTX (o). (B) hibition of bungarotoxin binding by carbamylcholine, however Results of an analogous experiment in which Ca2+-deficient buffer (Fig. 4). H2-HTX (1 1AM) causes a 10-fold shift in the KD for 1 was used for the preincubation. carbamylcholine from 14 to 1.4 ,vM. The shift in binding curves Competition experiments were carried out by incubating (Fig. 4) parallels the shift in the concentration against de- muscle cell cultures for 30 min with 3 nM radioactive bungar- sensitization curves (Fig. 2), suggesting strongly that H2-HTX otoxin and various concentrations of the ligand to be tested. increases the affinity of the desensitized form of the receptor Fig. 4 shows titration curves of the inhibition of bungarotoxin for carbamylcholine and thereby enhances desensitization. binding by carbamylcholine in the absence and presence of In order to determine whether H2-HTX also influences the constant concentrations of H2-HTX. In the absence of his- binding of other acetylcholine receptor ligands, similar titrations trionicotoxin, the KD for carbamylcholine was 14 AM. This were carried out in which bungarotoxin binding was inhibited value is in close agreement with previously published data (9) by the agonists acetylcholine, nicotine, and decamethonium and is much lower than the concentration of carbamylcholine and by the antagonists d-tubocurarine and gallamine. The re- required for half-maximal activation of receptor-dependent sults are presented in Table 1. With the four agonists tested, 1 Na+ transport (400 AM, ref. 8). Binding experiments involve AM H2-HTX caused a decrease of the concentration required long incubation with carbamylcholine, and therefore desensi- for 50% inhibition by a factor of 8.5 to 25. With the two an- tization of the receptor must occur during these experiments. tagonists, however, 1 ,uM H2-HTX did not cause a significant Comparison of these data with the data of Fig. 2 indicates that change in the inhibition curve. Thus, H2-HTX causes an in- the binding experiments measure the affinity of the desensitized crease in affinity of the acetylcholine receptor for agonists but form of the receptor for carbamylcholine. not for antagonists. Since only agonists cause desensitization, Since H2-HTX caused a shift in the concentration curve for these results provide further support for the view that H2-HTX desensitization (Fig. 2), it was of interest to study the effect of increases the affinity of the desensitized form of the receptor H2-HTX on carbamylcholine binding in '25I-labeled a-bun- for agonists. garotoxin competition experiments. Binding of bungarotoxin Previous work with the acetylcholine receptor of Torpedo was inhibited 50% by 20 AM H2-HTX (data not shown, com- electroplax has described an increase in affinity for agonists pare values on ordinate of Fig. 4). Concentrations of H2-HTX during incubation with agonists (16). In order to compare the (1 AM) that cause complete inhibition of receptor-dependent rate of the affinity increase with the rate of desensitization, we

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8 7 6 5 4 3 20 -Log molarity carbamylicholine c FIG. 4. Inhibition of (aX-BGT) ['251]monoiodo-ca-bungarotoxin 0 10 20 0 10 20 binding by carbamylcholine in the absence and presence of H2-HTX. Min Muscle cells were incubated (for 30 min) with 3 nM ['t5ljmonoiodo- ca-bungarotoxin and the indicated concentrations of carbamylcholine FIG. 5. Time course of [1251]diiodo-cy-bungarotoxin binding in (0) or carbamylcholine plus 0.1 (A), 1 (0), or 10 ,uM (0) H2-HTX. the absence and presence of inhibitors. (A) Muscle cells were incu- The inhibition by 100 ,uM H2-HTX is indicated on the ordinate (-). bated for the indicated times with buffer 1 containing 9 nM [1251]- Nonspecific binding of bungarotoxin has been subtracted. A + at the diiodo-a-bungarotoxin alone (0) and in the presence of 40 pM car- midpoint of the curves (Figs. 1, 2, and 4) indicates the concentration bamylcholine (A) and 5,uM carbamyicholine plus 1 AM H2-HTX (0) giving 50% of the maximum effect equivalent to the apparent disso- or 3 mM d-tubocurarine (0) (nonspecific binding). (B) Results of an ciation constant. analogous experiment in which Ca2+-deficient buffer 1 was used. Netirobiology: Burgermeister et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5757

have measured the initial rate of bungarotoxin binding in the 80- absence and presence of carbamylcholine and H2-HTX (Fig. .0 1.8 mM Ca2+ 5). During the first 15 min of incubation, total as well as non- -5 70 specific binding of 9 nM 1[25Ildiiodo-a-bungarotoxin to the -C cultures increased at a constant rate. When either 40 1M car- 0. bamylcholine or 5 AiM carbamylcholine plus 1 ,uM H2-HTX was a,= ) 50- present, the rate of bungarotoxin binding decreased gradually ~~No Ca'+ from an initial high value to a lower one which remained con- C40 stant (Fig. 5A). These results show that in cultured muscle cells, 30 as in electroplax (16, 17), exposure to carbamylcholine causes OI I an increase in affinity for carbamylcholine. Carbamylcholine 0 5 10 15 (5 AM) or 1 jiM H2-HTX, if individually present during the Min incubation, does not inhibit the binding of bungarotoxin (data FIG. 6. Ca2+-dependent change of receptor affinity in the pres- not shown). Therefore, H2-HTX enhances the ability of car- ence of carbamylcholine and H ,-HTX. Data from Fig. 5 have been used to plot percent inhibition of labeled bungarotoxin binding as a bamylcholine to cause the observed increase in affinity. When function of time. Inhibition )V 40 ,uMcarbamylcholine (A) or 5 M an analogous experiment was carried out with Ca2±-deficient carbamylcholine plus 1 pM H)-HTX (0) was determined in buffer buffer 1 (Fig. 5B), the same constant rates of total and non- 1(1.8 mM Ca2') and in Ca2'-deficient buffer 1, as indicated in the specific binding of bungarotoxin were observed. Under these figure. conditions, however, 40 jiM carbamylcholine or 5 puM carbamylcholine plus 1 yM H2-HTX did not cause a gradual (Figs. 2 and 3) is markedly reduced. Thus, 20 pM carbamyl- change of the bungarotoxin binding rate. When the percentage choline, a concentration that caused only negligible desensiti- of inhibition of the specific binding of bungarotoxin is plotted zation within 20 min, produced a 70% reduction of the Na+ as a function of time (Fig. 6), the effect of Ca2+ is clearly transport activity when H2-HTX was present (Fig. 3A). The demonstrated. In the presence of 1.8 mM Ca2 , the inhibition desensitization produced by carbamylcholine and H2-HTX by 40 pM carbamylcholine or by 5 pM carbamylcholine plus showed a marked dependence on the presence of Ca2+; in the 1 pM H2-HTX increased from 33 to 64 and 71%, respectively, absence of Ca2+, given concentrations of carbamylcholine, within 15 min. In the absence of Ca2 , the inhibition by the either alone or in combination with H2-HTX, caused much less same effectors remained at a level of 30-37% during the same desensitization than in the presence of 1.8 mM Ca2+. time period. The time course of the affinity increase, the en- The effects of H2-HTX on the binding of acetylcholine re- hancement of the affinity increase by H2-HTX, and the Ca2+ ceptor ligands, as shown by inhibition of labeled c-bungarotoxin dependence of the affinity increase are all consistent with the binding, can also be related to desensitization. Exposure of the conclusion that the increased affinity for carbamylcholine is receptor to agonists causes an increase in the affinity for agonists due to the formation of a high-affinity desensitized form of the (refs. 16 and 17, and Fig. 5). The time course and Ca2+ de- receptor. The results show that H2-HTX enhances this process pendence of this affinity increase are similar to the time course by increasing the affinity of the desensitized form of the re- and Ca2+ dependence of desensitization. In addition, our results ceptor for agonists. show that the concentration of carbamylcholine required to cause desensitization is one-tenth the concentration of car- DISCUSSION bamylcholine necessary to activate receptor-dependent Na+ transport. These observations indicate that the affinity for Cultured muscle cells have provided a suitable system for agonists increases during desensitization and that the desensi- studying the effects of H2-HTX on the nicotinic acetylcholine tized form of the receptor has 10-fold greater affinity for ago- receptor-ionophore complex. H2-HTX inhibited the car- nists than the active form. H2-HTX enhances desensitization bamylcholine-stimulated Na+ transport with a K, of 0.2 pM by causing an increase in the affinity of the desensitized form (Fig. 1). H2-HTX inhibits the depolarization produced by of the receptor for agonists and thereby stabilizing the de- carbamylcholine on the isolated electroplax of Electrophorus sensitized form. electricus with a K, of 0.8 pM (5). Eldefrawi et al. have found This mechanism for the action of H.-I-HTX is consistent with that 1 3HjH12-HTX binds to acetylcholine receptor-enriched the earlier electrophysiologic studies of Albuquerque et al. (2). membranes from Torpedo ocellata with a dissociation constant These authors showed that H.?-HTX had no effect on the end- K1) of 0.4 pM (7). In view of the differences in methods, there plate potential produced by a single pulse of acetylcholine but is good agreement among these values. reduced the endplate potential progressively during repetitive The inhibition of carbamvlcholine-dependent 22Na+ uptake pulses (figure 6 of ref. 2). These results are consistent with the bv H2-HTX is of a noncompetitive type. Noncompetitive in- conclusion that the principal effect of H2-HTX is to enhance hibition has also been reported by Kato and Changeux (5). The desensitization. binding of radioactive (Y-bungarotoxin to the acetylcholine The action of H2-HTX can be described on the basis of a receptor is inhibited by H2-HTX only at 100 times higher model of desensitization proposed by Katz and Thesleff (19) concentration than that of the inhibition of 22Na+ uptake. These and expanded by Rang and Ritter (20). results indicate that the blocking action of H2-HTX on neuromuscular transmission is caused by its interaction with a site A + R KD AR different from the acetvlcholine recognition site of the ace- resting active tylcholine receptor. This has also been concluded from results Ca2" of earlier investigations (2, 5, 6). Several results of the present investigation demonstrate di- H KD'I rectly an effect of H2-HTX on the desensitization of the ace- A + R' * AR' tylcholine receptor. Exposure to carbamylcholine has been desensitized S| K~ H2 HTX shown to cause rapid desensitization of the receptor Na+ A2-HTX tr transport activity (8, 15, 18). In the presence of H2-HTX, the HHT.-HXKD. AR' -H,-HTX A+R--H-HTX concentration of carbamylcholine required for desensitization 2 desensitized 5758 Neurobiology: Burgermeister et al. Proc. Natl. Acad. Sci. USA 74 (1977) In the model, A and R are symbols for the agonist and the ace- 1. Daly, J. W., Karle, I., Myers, C. W., Tokuyama, T., Waters, J. A. tylcholine receptor-ionophore complex. AR contains the & Witkop, B. (1971) Proc. Natl. Acad. Sci. USA 68, 1870- ionophore in an open state, whereas in AR' and AR"-H2-HTX, 1875. it is closed. As indicated by the increase of receptor affinity in 2. Albuquerque, E. X., Barnard, E. A., Chiu, T. H., Lapa, A. J., Dolly, J. O., Jansson, S.-E., Daly, J. W. & Witkop, B. (1973) Proc. the presence of the agonist (carbamnicholine), the dissociation Natl. Acad. Sci. USA 70,949-953. constant for the desensitized form, AR' is smaller than for the 3. Glavinovic, M., Henry, J., Kato, G., Krnjevic, K. & Puil, E. (1974) active form, AR, KD' < KD. H2-HTX binds to the desensitized Can. J. Physiol. Pharmacol. 52, 1220-1226. complex, AR'. The complex thus formed (AR"-H2-HTX) is also 4. Lapa, A. J., Albuquerque, E. X., Sarvey, J. M., Daly, J. & Witkop, a desensitized state, and contains A and H2-HTX bound si- B. (1975) Exp. Neurol. 47, 558-580. multaneously at separate sites. Our results show that KD" < KD' 5. Kato, G. & Changeux, J.-P. (1976) Mol. Pharmacol. 12, 92- < KD. This increase in the affinity for agonists is sufficient to 100. account for inhibition of receptor-dependent Na+ transport by 6. Dolly, J. O., Albuquerque, E. X., Sarvey, J. M., Mallick, B. & H2-HTX due to enhancement of densensitization. Barnard, E. A. (1977) Mol. Pharmacol. 13, 1-14. Eldefrawi et al. (7) have recently reported the separation of 7. Eldefrawi, A. T., Eldefrawi, M. E., Albuquerque, E. X., Oliveira, A. C., Mansour, N., Adler, M., Daly, J. W., Brown, G. B., Bur- acetylcholine- and HI2-HTX-binding proteins in a solubilized germeister, W. & Witkop, B. (1977) Proc. Natl. Acad. Sci. USA extract from Torpedo ocellata electric organ membranes. It was 74, 2172-2176. suggested, from these findings, that the HTX-binding protein 8. Catterall, W. A. (1975) J. Biol. Chem. 250, 1776-1781. might represent the ion conductance modulator or ionophore 9. Vogel, Z., Sytkowski, A. J. & Nirenberg, M. W. (1972) Proc. Natl. associated with the acetylcholine receptor. Since a tight cou- Acad. Sci. USA 69, 3180-3184. pling and mutual interaction of the acetvlcholine receptor and 10. Cahn, R. D., Coon, H. G. & Cahn, M. B. (1967) in Methods in ionophore has to be assumed, binding of H2-HTX to the iono- Developmental Biology, eds. Wilt, F. H. & Wessels, N. K. (T. Y. phore could cause stabilization of a desensitized form of the Crowell Co., New York), p. 525. 11. Fischbach, G. D. (1972) Dev. Biol. 28, 407-429. receptor in accordance with the model proposed here. A direct 12. Catterall, W. A. & Nirenberg, M. (1973) Proc. Natl. Acad. Sci. interference of H2-HTX with the ion transport of the ionophore USA 70, 3759-3763. cannot be ruled out on the basis of the present data. There is, 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. however, no experimental evidence in support of this mecha- (1951) J. Biol. Chem. 193, 265-275. nism of action. 14. Manthey, A. A. (1966) J. Gen. Physiol. 49, 963-976. 15. Sugiyama, H., Popot, J.-L. & Changeux, J.-P. (1976) J. Mol. Biol. 106, 485-496. Note Added in Proof. After submission of this manuscript, Elliot and 16. Weiland, G., Georgia, B., Wee, V. T., Chignell, C. F. & Taylor, Raftery (21) reported studies on the inhibition of (Y-bungarotoxin P. (1976) Mol. Pharmacol. 12, 1091-1105. binding by HTX and carbamylcholine in membrane fragments from 17. Weber, M., David-Pfeuty, T. & Changeux, J.-P. (1975) Proc. Natl. Torpedo electroplax. In contrast to our results with intact chick muscle Acad. Sci. USA 72,3443-3447. cells, these authors found no effect of HTX on bungarotoxin binding 18. Popot, J.-L., Sugiyama, H. & Changeux, J.-P. (1976) J. Mol. Biol. in the presence or absence of carbamylcholine and concluded, there- 106, 469-483. fore, that HTX had no effect on desensitization. Kato and Changeux 19. Katz, B. & Thesleff, S. (1957) J. Physiol. (London) 138, 63- (5) have, however, reported that HTX did increase the affinity of 80. acetylcholine for the receptor in membrane preparations from Torpedo 20. Rang, H. P. & Ritter, J. M. (1970) Mol. Pharmacol. 6, 357- electroplax, in complete agreement with our results in chick muscle 382. cells. The inability of Elliott and Rafterv to detect.such effects in their 21. Elliot, J. & Raftery, M. A. (1977) Biochem. Biophys. Res. Com- membrane preparation requires further investigation. mun. 77, 1347-1353.