Affinity labeling of the acetylcholine-receptor.

The monocellular electroplax of Electrophorus is a preparation particularly well suited for the study of the mechanism of action of specific ligands on the electrical parameters of an excitable membrane.' In the presence of low concentrations of compounds possessing a quaternary nitrogen function like acetylcholine (ACh), carbamylcholine (Carb), phenyltrimethylammonium (Pta), or decamethonium (Deca), the membrane potential of the cell decreases from -75 :i 15 millivolts (mV) in the resting cell to a steady-state value which depends on the nature and the concentration of the compound but is never lower than -15 4- 5 mV at saturating levels of the ligand. The effect of these receptor activators is specifically blocked by other quaternary nitrogen-containing compounds which will be referred to as receptor inhibitors like d-tubocurarine (d-Tubo), flaxedil, and several others, which stabilize the membrane potential at its resting value. All of them are postulated to bind to the same site on the membrane-the ACh-receptor site.2 In order to account for the remarkable specificity of recognition involved in the action of these ligands, the ACh-receptor site was further postulated to be part of a macromolecule, presumably a protein, and compared with the active center of an enzyme2 or the regulatory site of an allosteric protein.3 On the basis of these analogies it is of interest to study the ACh-receptor site with some of the methods which provided fruitful information about the amino acid side-chains constituting the active centers of enzymes and antibodies. In an affinity-labeling experiment4 a reagent is used which (1) exhibits a high affinity for the binding site; (2) forms an irreversible, covalent bond with certain amino acid residues of the site. This technique has been used to label specifically the active sites of several enzymes5 and antibodies4' 6 and it has been suggested as a means of