Proc. Nati. Acad. Sci. USA Vol. 75, No. 2, pp.,784-788, February 1978 Biochemistry

Alteration of the physicochemical properties of triphosphoinositide by nicotinic ligands (neuromuscular blockers/hydrophobicity/phosphatidylinositol bisphosphate) TAE MOOK CHO, JUNG SOOK CHO, AND HORACE H. LOH Langley Porter Neuropsychiatric Institute and Department of Pharmacology, University of California, San Francisco, California 94143 Communicated by Choh Hao Li, December 2,1977

ABSTRACT The concentrations of nicotinic drugs required from cerebral cortex by De Robertis et al. (7). Chemical de- to effect a 50% transfer of triphospho[3H]inositide from an terminations confirmed that P3inositide is present in this re- aqueous phase to a nonaqueous phase closely approximated ceptor fraction and suggested that P3inositide is the major their concentrations for both in vivo neuromuscular blocking activity and binding to purified nicotinic receptors, and corre- component responsible for the binding of nicotinic ligands lated well (correlation coefficient = 0.95) with their biological (6). activities measured by other workers in an Electrophorus The present study describes selective alterations in the electroplax preparation. The triphospho[3Hjinositide transfer physicochemical properties of P3inositide on interaction with induced by nicotinic ligands was dependent on the lipid con- various nicotinic ligands. The alterations have been shown to centration and was potentiated by Ca +. The affinity constants be related to the neuromuscular blocking activity of these li- of 45Ca2+ for triphosphoinositide were similar to those for the purified nicotinic receptor. These and other findings suggest gands. The results suggest that P3inositide may function as a the possibility that triphosphoinositide (phosphatidylinositol binding component of the nicotinic cholinergic receptor. is a site of the nicotinic bisphosphate) binding receptor. MATERIALS AND METHODS Recently, Cavallito (1) and others (2, 3) have reported that Chemicals. P3inositide was isolated by the method of Michell electrostatic bond formation between the cationic portion of et al. (8), further purified using triethylaminoethyl-cellulose nicotinic ligands and the anionic site of the cholinergic receptor column chromatography, and identified by the method of is essential for pharmacological activity. Cavallito further Gonzalez-Sastre and Folch-Pi (9), using thin-layer chroma- suggested that the highly charged phosphate macromolecules tography and visualization by iodine, which yielded a single may be more closely related to the nicotinic receptor than are spot with an RF value the same as the reported one. The P3- monovalent anions such as carboxylate, phosphate, and sulfate inositide spot was removed and the phosphate content was (4). From structural considerations of triphosphoinositide determined by Bartlett's method (10). On the basis of the (Psinositide; phosphatidylinositol bisphosphate), a membrane phosphate determination, the P3inositide was over 95% pure. acidic lipid with a maximum of five negative charges, it is P3[3H]inositide (specific activity 3 Ci/mmol) was prepared by theoretically possible that the molecule could interact with a C. T. Peng (School of Pharmacy, University of California, San variety of structurally unrelated nicotinic ligands. For example, Francisco) at the Lawrence Laboratories (Berkeley, CA). the.cationic nitrogen and carbonyl oxygen of a drug such as 45CaCl2 (20 Ci/g) was obtained from New England Nuclear carbachol could bind to an anionic oxygen atom in the phos- (Boston, MA). phate group and to a hydroxyl group of the inositol ring, re- The following chemicals were purchased from Sigma spectively. Similarly, the distance between the two cationic Chemical Co.: d-tubocurarine chloride, decamethonium nitrogen atoms in the hexamethonium molecule is similar to chloride, trimethylbenzylammonium chloride, succinylcholine the distance between two anionic oxygen atoms of the phos- chloride, hexamethonium chloride, carbachol chloride, pilo- phates in the 4 and 5 positions of the inositol ring. Furthermore, carpine, methacholine chloride, (+)-muscarine chloride, 5- the distance between the quaternary nitrogen atoms in cho- hydroxytryptamine, dopamine, epinephrine, norepinephrine, linergic ligands such as decamethonium, succinylcholine, gal- and histamine. Nicotine, 1,1-dimethylphenylpiperazonium lamine, and d-tubocurarine matches the distance between the iodide, and lobeline were purchased from K & K Laboratories; two anionic oxygen atoms of the phosphate group in positions gallamine triethiodide from Davis and Geck, American Cy- 1 and 4 or 1 and 5 of the inositol ring. These theoretical con- anamid Co.; and mecamylamine from Merck, Sharp and siderations are supported by experimental data showing that Dohme Research Laboratories. a-Bungarotoxin was a gift from P3inositide binds nicotinic agonists and antagonists with high C. C. Chang of National Taiwan University (Taipei, Republic affinities, and that the dissociation constants obtained are of China). comparable to those reported for the same agents binding to Preparation of P3inositide Micelles. P3[3H]inositide was purified nicotinic receptor (5). Of the membrane acidic lipids mixed with unlabeled P3inositide in a mixture of chloroform examined, only P3inositide selectively binds di[14C]methyl- and methanol (2:1) and the solution was then taken down to d-tubocurarine ([14CJDMTC), and the chromatographic be- dryness on a rotary vacuum evaporator, after which the dried havior of the [14C]DMTC-PNinositide complex on a Sephadex P,3inositide powder was suspended in 5 ml of Tris-HCI buffer LH-20 column (6) is identical to the behavior of the DMTC (2 mM, pH 7.4) and allowed to swell for an hour. The P3inosi- complex formed with the proteolipid receptor fraction isolated tide was sonicated for 1 min and then diluted with the same The costs of publication of this article were defrayed in part by the Abbreviations: P3inositide, triphosphoinositide (phosphatidylinositol payment of page charges. This article must therefore be hereby marked bisphosphate); DMTC, dimethyl-d-tubocurarine; IC50, concentration "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate required to induce a 50% transfer of P3inositide from aqueous to this fact. nonaqueous phase. 784 Downloaded by guest on September 26, 2021 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 75 (1978) 785 buffer to give an appropriate final P3inositide concentra- tion. 'or Interface P3[3H]inositide Transfer Studies. Five tenths milliliter of 9 the P3[3H]inositide micelle preparation was added to 0.5 ml of Tris buffer containing drug and/or cation at room temperature. 8 This aqueous solution was mixed with 1 ml of heptane on a Vortex mixer (speed setting at 5) for 1 min and the mixture was 0 7 centrifuged at 1500 X g for 10 min. A 0.5-ml aliquot of the x aqueous phase mixed with 5 ml of Scintiverse cocktail solution E6 (Fisher Scientific Co.) was assayed in a Beckman liquid scin- tillation counter to determine P3[3H]inositide activity. The 5 radioactivity in the nonaqueous phase (heptane plus interphase) 0. was determined by subtracting the radioactivity in the aqueous 0o phase from the total amount added. The percent of drug-in- I duced P3[3H]inositide transferred from the aqueous phase to 31 K\ie Heptane phase the nonaqueous phase was calculated by using the following 0 ° ~Oto. equation: at---la 2 F 4__hi % of P3[3H]inositide transferred I Aqueous phase radioactivity without - radioactivity with drug I I I I I I I I drug in aqueous phase in aqueous phase x 100 1 2 3 4 5 6 7 8 9 10 radioactivity without drug [Decamethoniuml, MM FIG. 1. Distribution of P3[3H]inositide in the presence and ab- in aqueous phase sence of decamethonium. The influence of various concentrations of 45Ca2+ Binding to P3inositide. One hundred milliliters of decamethonium on the distribution of P3[3H]inositide between the 45Ca2+ was mixed with 100 ml of the nonra- aqueous phase, the heptane phase, and the interface was determined (2 puM) prepared by measurement of the radioactivity in the buffer and heptane com- dioactive P3inositide micelles (20 jg/ml). An aliquot of the ponents. The amount ofP3[3H]inositide at the interface was indirectly mixture (0.5 ml) was added to a 0.5-ml solution containing deduced by calculations, subtracting the measured radioactivities various concentrations of unlabeled calcium or neuromuscular from the total. blocking agents and the aqueous solution was mixed with 1 ml of heptane on a Vortex mixer (speed at 5) for 1 min. The mix- K1 = 2.7 AtM, K2 = 25 MM, and K3 = 350 AM. These data ture was centrifuged at 1500 X g for 10 min and the radioac- suggest that in the presence of 100 AM calcium ion only the tivity of 45Ca2+ in the lower phase was determined by the same binding sites with the two lower dissociation constants (K1 and method described for the P3[3H]inositide transfer studies. 00 r- RESULTS Distribution of P3[3H]inositide. The radioactive P3inositide A'S in both the heptane and aqueous phases was determined in the presence and absence of decamethonium, and the amount of 8C /O labeled P3inositide at the interphase was obtained by sub- of. tracting the measured radioactivities from the total added. As shown in Fig. 1, the P3[3H]inositide in the aqueous phase de- 4O~0) creased with increasing concentration of the decamethonium (A added, while the radioactivity in the nonaqueous phases in- a) 60 creased, particularly in the interphase. c La 3+ ICa2t 0' Effects of Calcium and Lanthanum Ions on P3[3H]inosi- 'a (a tide Transfer. Fig. 2 shows that both calcium and lanthanum, *0 a calcium antagonist, promoted P3[3H]inositide transfer from 0 the aqueous to the nonaqueous phase in a concentration-de- 40I pendent manner. The concentration of lanthanum required to I- 0/ induce a 50% transfer from the aqueous to the nonaqueous 4 phase (IC50) was approximately 250 times less than that of calcium. The P3[3H]inositide transfer was also determined with 20 increasing concentrations of decamethonium and d-tubocu- 0 rarine in the presence and absence of 100 AM calcium ion, 0l which by itself did not significantly induce the transfer (Fig. 3). Calcium caused a 2-fold increase in the P3[3H]inositide transfer induced by both drugs and appeared to change the L- shape of the transfer-concentration curves from sigmoid to 10 10 5 104 10-, hyperbolic. To elucidate further the effect of calcium on [Cation], M transfer induced by nicotinic FIG. 2. Transfer of P3[3H]inositide induced by Ca2+ and La3+. P3[3H]inositide ligands, 45Ca2+ Inorganic ion-induced transfer of labeled P3inositide (0.2 gAg/ml) from binding to P3inositide was determined and [Ca2+hboUnd/ the aqueous to the nonaqueous phases was determined from mea- [Ca2+]free was plotted against [Ca2+]bound As shown in Fig. 4, surement of the change of radioactivity in the aqueous phase after calcium had at least three dissociation constants for P3inositide: addition of the ions as described in Materials and Methods. Downloaded by guest on September 26, 2021 786 Biochemistry: Cho et al. Proc. Nati. Acad. Sci. USA 75(1978)

100 100 A "IOS _ PR' -- 8 0 80 Ci+ O/ /C2+ +Ca-2+ -'2. :2 X +Ca " o-/0Ca 4) .U 60

4) c! _'A, w 40 I I 0) 0 C 4- 4 - I o' I 20 I 0 /g 2 4 6 8 10 2 3 4 IL [Decamethonium], JM [d-TubocurarineJ, ,M FIG. 3. P3[3H]inositide transfer induced by decamethonium and 2 3 4 5 d-tubocurarine: effects of 100 AM CaC12. Increasing concentrations [Gallaminel, }AM of decamethonium and d-tubocurarine in the presence and absence of 100AM CaC12 were used to induce the transfer of 0.2 Ag/ml of tri- FIG. 5. Gallamine-induced P3P3H]inositide transfer at different tiated P3inositide from the aqueous to the nonaqueous phase. (A) P3inositide levels. P3[3H]inositide at 0.2 and 5.0 Mg/ml was mixed with Decamethonium; (B) d-tubocurarine. increasing concentrations ofgallamine in order to induce transfer from the aqueous to the nonaqueous phases. K2) may be saturated. Additional experiments were performed ligands, noncholinergic neurotransmitters, and inorganic cat- to on study the effect of d-tubocurarine and gallamine 45Ca2+ ions-were tested for their ability to affect the Ps[3H]inositide (0.5 ,uM) binding to (data not shown). At the concentrations used transfer at two different concentrations of P3inositide in the in Fig. 3, neither drug inhibited the binding of 45Ca2+ to P3- presence and absence of calcium ion. Fig. 6 shows the con- inositide, indicating that the calcium-binding sites with higher centration-response curves for the nicotinic ligands determined affinities (K1 and K2) may be different from the nicotinic from the data obtained with P3inositide at 0.2 ,gg/ml in the binding sites. presence of 100( ,M calcium ion. The slopes were steep and the Effects of P3inositide Concentration on P3[3H]inositide curves were parallel. The IC50 values were determined from Transfer. The effect of P3inositide concentration on drug- the curves by extrapolation and the results are summarized in induced P3[3H]inositide transfer was also studied at two dif- Table 1. Of the experimental conditions tested, 100 ,uM of Ca2+ ferent P3inositide concentrations (0.2 and 5 ,ug/ml) in the and 0.2 tig/ml of P3inositide required the lowest concentration presence of gallamine. As shown in Fig. 5, at the higher P31. of ligands to induce the P3[3H]inositide transfer. Under these inositideconcentration,the1IC5o of gallamine was 3 times higher conditions, nicotinic ligands were much more potent than either than at the lower P3inositide concentration, and the transfer- the muscarinic ligands or the noncholinergic neurotransmitters, concentration curve was a to a more shifted from hyperbolic and their ICso values were about one-third as high as their sigmoid shape. This effect of P3inositide concentration was also corresponding in vitro biological activities and apparent af- seen with other cholinergic ligands (Table 1). finities as reported by Cohen and Changeux (11). The IC,0 Potency, Specificity, and "Biological Correlation." Various values of the nicotinic compounds appeared to correlate more compounds-including nicotinic and muscarinic cholinergic with their neuromuscular blocking activities, such as those represented by the Electrophorus receptor, than with their 0.20 r ganglionic blocking activities. For example, potent neuro- muscular blockers such as a-bungarotoxin, pancuronium, al- 0.18 curonium, d-tubocurarine, and gallamine were very effective in transferring P3[3H]inositide, while ganglionic blockers such 0.16

1 1 1 2 3 4 5 6 7 8 9 12 o0.14 - 80 /

- n 0.12 K=2.7M 70_

U ax

n 0.10 - C6 L._ 0 0~~~0 + 0.08 \ . 50

30 0.06 - 40 I40

0.04 - K2 25uMM II 10-7 10-6 lo-, 10-4 0.02 [Drug], M FIG. 6. Concentration-response curves for P3[3H]inositide I I II I transfer induced by nicotinic ligands. Dose-response curves for a 0.2 0.4 0.6 0.8 1.0 1.2 1.4 variety of nicotinic ligands capable of inducing the transfer of tritiated Calcium bound, nmol P3inositide (0.2 Mg/ml) from the aqueous to the nonaqueous phases FIG. 4. Scatchard plot of 45Ca2+ binding. P3inositide at 5Mug/mi were determined. The data points are the mean of triplicate samples and various concentrations ofCaCl2 were used. It should be mentioned in two separate experiments. 1, a-Bungarotoxin; 2, d-tubocurarine; that although most of free P3inositide occurred in the aqueous phase 3, gallamine; 4, decamethonium; 5, mecamylamine; 6, lobeline; 7, di- under these conditions, calcium binding was observed mostly at the methylphenylpiperazonium; 8, trimethylbenzylammonium; 9, nico- interface due to the small amount of P3inositide. tine; 10, succinylcholine; 11, hexamethonium; 12, carbachol. Downloaded by guest on September 26, 2021 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 75 (1978) 787

Table 1. Concentration of various compounds required to induce P3[3H]inositide transfer by 50% (IC50) IC50, AsM ICarbachol P3inositide P3inositide (0.2 mg/ml) (5.0 jg/ml) 10- Compound -Ca2+ +Ca2+ -Ca2+ +Ca2+ Nicotinic ligands a-Bungarotoxin 0.5 0.18 1.2 0.6 MX lo-6 Pancuronium 0.4 0.25 Alcuronium 0.35 0.25 - * Gallamine Dimethyl-d-tubo- 0 Tubocurarine curarine 0.60 0.30 IJ d-Tubocurarine 1.1 0.50 3.0 1.2 10 6 10 5 10-4 10-3 Gallamine 1.6 0.80 4.5 1.8 IC50, M Decamethonium 3.0 2.1 7.6 3.0 FIG. 7. Correlation of the IC50 values of P3[3H]inositide transfer Mecamylamine 6.0 4.6 15 7.4 with the in vitro potency of various ligands. The IC50 values of these Dimethylphenylpipera- nicotinic drugs were taken from Table 1 and the values of Kapp (ap- zonium 22 15 35 25 parent dissociation constant) were obtained from the isolated Trimethylbenzyl- monocellular electroplaque by Cohen and Changeux (11). ammonium 42 26 80 46 Nicotine 92 50 160 90 ligands correlate closely with their in vivo neuromuscular Lobeline 19 10 40 20 blocking activities. Succinylcholine 84 120 150 130 The precise nature of the interaction between cholinergic Hexamethonium 180 130 300 210 ligands and P3inositide in micellar form is not clear, but com- Carbachol 250 210 550 350 plex formation appears to alter the physicochemical properties Muscarinic ligands of the lipid molecule to result in increased hydrophobicity (here Pilocarpine 110 100 200 150 defined as tendency to leave the aqueous phase). The IC50 Methacholine 150 150 300 200 values determined are not solely dependent on the affinity (W)-Muscarine 250 210 3000 2000 (dissociation constant) of the ligand for P3inositide, but also reflect the apparent change in physicochemical properties of Neurotransmitters the complex formed. Thus, for any given ligand, the ratio of KD 5-Hydroxytryptamine - 130 to ICWo is a measure of the relative hydrophobicity of the li- Dopamine - 230 - gand-P3inositide complex. In the case of the stabilizing Epinephrine 500 blocking agent d-tubocurarine, with a KD of 0.2 ,uM (unpub- Norepinephrine - 800 lished data) this ratio is approximately 0.4. However, with Histamine 1100 succinylcholine, a depolarizing blocking agent (KD 1.6 ,uM), Cations the ratio is much lower (0.013), suggesting that at ligand con- CaCl2 350 150 centrations close to the dissociation constant the succinylcho- LaCl3 0.9 1.1 - line-P3inositide complex is less hydrophobic than the complex IC50 values were obtained from plots of percent of P3[3H]inositide of d-tubocurarine with P3inositide. At concentrations of suc- transferred vs. the log concentrations of the various compounds by cinylcholine closer to the IC50 value, the icomplex with P3- extrapolation at two P3inositide concentrations and in the presence inositidebecomes more hydrophobic. Suchbehavior is consistent or absence of 100 ,uM CaCl2. Data points are the mean of triplicate with ion-pair theory, which states that the formation of intimate determinations in two separate experiments. ion pairs is dependent on the concentration of the cation in a given concentration of the anion (13, 14). The formation of two as hexamethonium and mecamylamine were relatively weak. different types of succinylcholine-P3inositide complex may However, it should be noted that mecamylamine, with an IC50 have relevance to our understanding of the biphasic effects of twice that of decamethonium, was only half as potent as depolarizing neuromuscular blocking agents and of discrimi- decamethonium in the phrenic nerve diaphragm preparation nation between stabilizing and depolarizing actions of the (12). The IC50 values of the nicotinic ligands were plotted nicotinic blocking agents. against the biological potencies obtained from the isolated It is well known that calcium ion increases the binding of the Electrophorus electroplax (Fig. 7). According to the method cholinergic ligands to their receptors (15, 16). We also observed of linear regression analysis, the data were fitted to the line calcium potentiation of P3[3H]inositide transfer induced by (correlation coefficient = 0.95). such ligands (Fig. 3). Because 100 ,uM CaCl2 was by itself not sufficient to transfer the Pa[3H]inositide from the aqueous to DISCUSSION the nonaqueous phase, the calcium potentiation appeared due From the molecular considerations as well as experimental to the enhancement of the affinity of the ligand to P3inositide. findings presented previously, we have suggested that P3inos- This has been confirmed and, as in the case of the nicotinic itide, a membrane acidic lipid, may be a binding component receptor, the binding of ['4C]DMTC and [3H]decamethonium in the nicotinic chloinergic receptor. Using the drug-induced to P3inositide was also enhanced by various concentrations of transfer of P3[3H]inositide from an aqueous to a nonaqueous calcium when the experiments were performed in Krebs- phase, we have determined the IC50 of various nicotinic and Ringer solution. However, in Tris.HCI buffer (pH 7.4), calcium muscarinic ligands, noncholinergic neurotransmitters, and enhanced the binding of [14C]DMTC to P3inositide, whereas cations, and we have found that the IC50 values of the nicotinic the binding of [3H]decamethonium was inhibited. Thus, cal- Downloaded by guest on September 26, 2021 788 Biochemistry: Cho et al. Proc. Natl. Acad. Sci. USA 75 (1978) cium discriminates the stabilizer from the depolarizer in the that physicochemical properties (i.e., hydrophobicity and hy- drug binding to P3inositide (T. M. Cho and H. H. Loh, un- drophilicity) of the ligand-receptor complex may be important published data). in the discrimination of the neuromuscular blocking agents Several lines of evidence appear to support a possible role for from the stimulating agents. P3inositide as a binding component of the nicotinic receptor: (i) The concentrations of nicotinic ligands required to induce 1. Cavallito, C. J. (1962) in Curare and Curare-like Agents, ed. De 50% transfer from the aqueous phase to the Reuck, A. V. S. (Little Brown & Co., Boston, MA), pp. 55-70. P3[3H]inositide 2. Bovet, D. (1972) in Neuromuscular Blocking and Stimulating nonaqueous phase (IC5o) not only were close to the concen- Agents, ed. Cheymol, J. (Pergamon Press, Oxford), Vol. 1, pp. trations required for the ligands' in vio neuromuscular 243-294. blocking activities and their binding to the purified nicotinic 3. Cheymol, J. & Bourillet, F. (1972) in Neuromuscular Blocking receptor, but also correlated well with their in vitro pharma- and Stimulating Agents, ed. Cheymol, J. (Pergamon Press, cological activities obtained from the isolated eel electroplax Oxford), Vol. 1, pp. 297-356. and reported by Cohen and Changeux (11). (ii) Calcium en- 4. Cavallito, C. J. (1967) Ann. N.Y. Acad. Sci. 144,900-912. hanced the ligand-induced transfer of P3[3H]inositide, and this 5. Cho, T. M. & Loh, H. H. (1975) Pharmacologist 17,254. cation has been shown to increase binding of nicotinic ligands 6. Wu, Y. C., Cho, T. M. & Loh, H. H. (1977) J. Neurochem. 29, to the and to increase of 589-592. cholinergic receptor (15, 16) binding 7. De Robertis, E., Fiszer, S., Pasquini, J. M. & Soto, E. F. (1969) J. [14C]DMTC and [3H]decamethonium to Psinositide.* The Neurobiol. 1, 41-52. dissociation constants for the Ca2+-Painositide complex were 8. Michell, R. H., Hawthorne, J. N., Coleman, R. & Karnovsky, M. almost identical to those for the purified nicotinic receptor- L. (1970) Biochim. Biophys. Acta 210,86-91. Ca2+ complex (15). (iii) De Jobertis et al. have shown binding 9. Gonzalez-Sastre, F. & Folch-Pi, J. (1968) J. Lipid Res. 9,532- of [14C]DMTC, a nicotinic antagonist, to the purified proteo- 533. lipid nicotinic receptor isolated from both cat brain (7) and 10. Bartlett, G. R. (1959) J. Biol. Chem. 234,466-468. electric eel (17). Such proteolipid receptors appear similar to 11. Cohen, J. B. & Changeux, J.-P. (1975) Annu. Rev. Pharmacol. detergent-extracted cholinergic protein receptors except for 15,83-109. their immunological properties (18, 19). Using the method of 12. Burn, J. H. & Seltzer, J. (1965) J. Physiol. (London) 179, 569- 576. De Robertis et al. to purify nicotinic cholinergic receptors, we 13. Gordon, J. E. (1975) in The Organic Chemistry of Electrolyte have reported the presence of P3inositide in this purified re- Solutions, ed. Gordon, J. E. (Wiley, New York), pp. 371-520. ceptor fraction and shown that the binding of [14C]DMTC is 14. Szwarc, M. (1972) in Ions and Ion Pairs in Organic Reactions, mainly, if not solely, due to its interaction with P3inositide in ed. Szwarc, M. (Wiley, New York), Vol. 1, pp. 1-24. this fraction (6). Furthermore, the chromatographic behavior 15. Chang, H. W. & Neumann, E. (1976) Proc. Natl. Acad. Sci. USA of the [14C]DMTC-P3inositide complex was identical to that 73,3364-3368. of the [14C]DMTC complex as reported by De Robertis et al. 16. Cohen, J. B., Weber, M. & Changeux, J.-P. (1974) Mol. Phar- (7). macol. 10, 904-932. On the basis of the present findings, it is tempting to speculate 17. De Robertis, E. & De Plazas, S. F. (1970) Biochim. Biophys. Acta 219,388-397. that neuromuscular blocking activity of the antagonist type may 18. Komentani, T., Ideda, Y. & Kasai, Y. (1975) Biochim. Biophys. be elicited via the formation of hydrophobic ligand-receptor Acta, 413, 415-424. complexes, thereby stabilizing the membrane with blockade 19. De Robertis, E., De Plazas, S. F. & De Carlin, M. C. L. (1976) of the ionic conductance. Furthermore, our findings indicate Nature 259,605. Downloaded by guest on September 26, 2021