Molecular Pharmacology, Vol 10, 904-932, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Effects of Local Anesthetics and Calcium on the Interaction of Cholinergic Ligands with the Nicotinic Receptor Protein from Torpedo marmorata

¹ Unité de Neurobiologie—Département de Biologie Moléculaire, Institut Pasteur, 75015 Paris, France

Studies are presented of the interaction in a physiological ionic environment of aromatic amine local anesthetics (prilocaine, lidocaine, and dimethisoquin) and Ca⁺⁺ with receptor-rich membrane fragments isolated from Torpedo electric organ. The environmentaly sensitive fluorophore 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane 2-trimethylammonium iodide (DNS-chol)interacts with two classes of sites in the membrane fragments: the cholinergic receptor site and secondary sites characterized by probe emission properties (_max) sensitive to the pharmacological nature (agonist or antagonist) of the cholinergic ligand bound to the receptor site. Fluorescence studies show that the local anesthetics cause an increase of affinity of the membrane-bound receptor for DNS-chol and for cholinergic ligands, both agonists and antagonists. The increase of affinity is not associated with a change of DNS-chol emission properties. At the same concentrations at which the anesthetics control receptor affinity, they also affect the fluorescence of DNS-chol bound to the secondary sites: the presence of local anesthetic causes a loss of the DNS-chol spectral properties characteristic of the binding of agonists to the receptor site. Local anesthetics also control the binding of [³H]acetylcholine to the membrane-bound receptor. In the absence of prilocaine the acetylcholine binding curve is slightly sigmoid (Hill coefficient, n_H = 1.4, half-saturation at 10 nM free acetylcholine). In the presence of 3 mM prilocaine there is a decrease of cooperativity and an increase of affinity (n_H = 1.0, half-saturation at 6 mM free acetylcholine). The concentrations at which the local anesthetics act on the membrane fragments are those at which they block the permeability response of Electrophorus electroplax upon addition to the bath of the agonist carbamylcholine. Fluorescence and radioactive ligand assays demonstrate that Ca⁺⁺ also causes an increase of receptor affinity for cholinergic ligands, but in a manner significantly different from that observed with local anesthetics. Solubilization of membrane fragments by detergent leads to changes in the binding properties of the receptor protein. On the membrane fragments the binding data for each agonist can be analyzed in terms of a homogeneous population of sites, while after solubilization heterogeneity of the binding constants appears. Prilocaine or Ca⁺ no longer affects the binding of acetylcholine to the solubilized receptor protein. The observed effects of local anesthetics and Ca⁺⁺ on the affinity of the cholinergic receptor are related to the phenomenon of receptor desensitization.

Note:
ACKNOWLEDGMENT We thank Professor P. Boquet for a gift of purified Naja -toxin; Drs. A. Menez, J. L. Morgat, and P. Fromageot, for its tritiation; Dr. C. Cazaux and his colleagues at the Institut de Biologie Marine, Arcachon, for supplying Torpedo; and Dr. P. Ascher, for a gift of SKF 525-A. Finally, we thank Drs. H. Buc and R. Sealock for their helpful discussions.

This article has been cited by other articles:

				S. E. Ryan and J. E. Baenziger A Structure-Based Approach to Nicotinic Receptor Pharmacology Mol. Pharmacol., February 1, 1999; 55(2): 348 - 355. [Abstract] [Full Text]

				J. Changeux, A Devillers-Thiery, and P Chemouilli Acetylcholine receptor: an allosteric protein Science, September 21, 1984; 225(4668): 1335 - 1345. [Abstract] [PDF]

				M. Raftery, M. Hunkapiller, C. Strader, and L. Hood Acetylcholine receptor: complex of homologous subunits Science, June 27, 1980; 208(4451): 1454 - 1456. [Abstract] [PDF]

				J.-P. Changeux, L. Benedetti, J.-P. Bourgeois, A. Brisson, J. Cartaud, P. Devaux, H. Grunhagen, M. Moreau, J.-L. Popot, A. Sobel, et al. Some Structural Properties of the Cholinergic Receptor Protein in Its Membrane Environment Relevant to Its Function as a Pharmacological Receptor Cold Spring Harb Symp Quant Biol, January 1, 1976; 40(0): 211 - 230. [Abstract] [PDF]