Molecular Pharmacology, Vol 10, 15-34, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Binding of Naja nigricollis [³H]-Toxin to Membrane Fragments from Electrophorus and Torpedo Electric Organs

II. Effect of Cholinergic Agonists and Antagonists on the Binding of the Tritiated -Neurotoxin

¹ Neurobiologie Moléculaire, Institut Pasteur, Paris XV^e, France

The effects of nicotinic effectors on the kinetics of association and dissociation and on the binding at equilibrium of a tritiated -neurotoxin from Naja nigricollis with membrane fragments purified from Electrophorus and Torpedo electric organs are studied. Increasing concentrations of nicotinic agonists and antagonists decrease the initial rate of [³H]-toxin binding to membrane fragments from both species. This rate becomes negligible at high concentrations of effectors. The "protection curves" obtained are compared with the binding curves of radioactive effectors to the same membrane fragments. In the case of Torpedo membrane fragments, the binding curve of [³H]-acetylcholine (in the presence of O,O-diethyl S-(-diethylamino)ethyl phosphorothiolate) is slightly sigmoid (n_H = 1.3); half-saturation occurs at 8 nM acetylcholine. [³H]-Decamethonium binds to one class of sites with K_D = 0.8 µM, and possibly to another class with lower affinity. The binding of both acetylcholine and decamethonium is competitively inhibited by d-tubocurarine (K_D = 0.2 µM) and completely displaced by -toxin. The numbers of decamethonium and acetylcholine binding sites on Torpedo membrane fragments are very close to the number of [³H]-toxin binding sites. The binding curves of decamethonium and acetylcholine can be superimposed on the "protection curves" of these two agonists against [³H]-toxin binding. The data are in terpreted on the basis of a mutual exclusion of cholinergic effectors and -toxin from a common site, which is identified as the nicotinic receptor site. Comparison of our protection data with the binding data of Kasai and Changeux [(1971) J. Membr. Biol., 6, 1-80] shows that the same result holds in the case of Electrophorus membrane fragments. The dissociation constants of a large spectrum of cholinergic effectors were determined by following the protection against [³H]-toxin. The pharmacological properties of the receptors from Electrophorus and Torpedo appear different. In the case of Electrophorus, the dissociation constants for all the agonists and antagonists coincide with the "apparent dissociation constants" measured in vivo on the electroplax. At equilibrium decamethonium and d-tubocurarine displace [³H]-toxin from Electrophorus membrane fragments; the data are in given domains of concentrations, compatible with the hypothesis of a mutual exclusion of -toxin and cholinergic effectors from the same binding site. However, higher concentrations of both ligands enhance the rate of dissociation of [³H]]-toxin from its membrane site.

Note:
ACKNOWLEDGMENTS We thank Professor P. Boquet for purification and a generous gift of pure -toxin; Drs. A. Menez, J.-L. Morgat and P. Fromageot for its tritiation; Professor P. G. Waser for the gift of muscarone; and the Laboratoire Roger Bellon for the gift of dimethisoquin and prilocaine. We thank Drs. R. L. Baldwin, H. Buc, J. B. Cohen, G. L. Hazelbauer, H. Lester, J. C. Meunier, R. W. Olsen, and H. Sealock for helpful criticism and suggestions and aid in the preparation of the manuscript. We thank Dr. J. Patrick for the privileged communication of a manuscript in publication.

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