Molecular Pharmacology, Vol 15, 294-312, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Identification of a Local Anesthetic Binding Site in Nicotinic Post-Synaptic Membranes Isolated from Torpedo marmorata Electric Tissue

¹ Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Studies are presented of the interaction of aromatic amine noncompetitive antagonists with the receptor-rich (post-synaptic) membranes isolated from Torpedo marmorata electric organ. When present at micromolar concentrations, methyl iodide quaternary derivatives of the potent non-competitive antagonists proadifen and dimethisoquin increase the affinity of the membrane-bound nicotinic receptor for [³H]acetylcholine and [¹⁴C]dimethyltubocurarine. The equilibrium binding of [¹⁴C]meproadifen (2-(diethylmethylamino)ethyl-2,2-diphenyl valerate) to the receptor-rich membranes is characterized by an ultracentrifugatiom assay. When all acetylcholine binding sites are occupied by the agonist carbamylcholine or the antagonist tubocurarine, [¹⁴C]meproadifen is bound with a dissociation constant K_D = 0.5 µM to a number of sites equal to one-quarter the number of -neurotoxin binding sites. That high affinity binding site is found in the receptor-rich membranes and not in other membrane fractions isolated from Torpedo electric organ. Other non-competitive aromatic amino antagonists including dimethisoquin, proadifen, and prilocaine displace [¹⁴C]meproadifen, as does perhydrohistrionicotoxin. It is concluded that there is a specific site of binding for the aromatic amine non-competitive antagonists in the isolated nicotinic post-synaptic membrane that is distinct from the site of binding of acetylcholine. Analysis of the interaction of [¹⁴C]meproadifen with the receptor-rich membranes in the absence of cholinergic ligands indicates that under these circumstances the ligand binds weakly to both the anesthetic binding site and the acetylcholine binding site (K_D = 5 µM). The functional significance of the specific binding site for the aromatic amine non-competitive antagonists is discussed in terms of its possible relation to the site of ion translocatiom and to the mechanism of receptor desensitization.

Note:
ACKNOWLEDGMENTS We are grateful for the expert technical assistance provided by Dan C. Medynski in the early stages of this investigation. We thank Drs. A. Menez, J. L. Morgat, P. Fromageot, and P. Boquet for a gift of the [³H]-neurotoxin of N. nigricollis; Prof. Y. Kishi for his gift of H₁₂-HTX, which he and his associates synthesized (26); Dr. B. Takman (Astra Pharmaceuticals) for a gift of prilocaine, lidocaine, and lidocaine ethylbromide; Dr. E. S. Rump (Smith, Kline, and French) for samples of dimethisoquin and proadifen. In addition we are grateful to Dr. C. Cazaux and his colleagues at the Institut de Biologie Marine, Arcachon, for supplying Torpedo and to L. Garibaldi, J. Dayton, and E. Kettle (New England Aquarium) for their maintenance.

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