Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anesthetics and histrionicotoxin

Highly purified subsynaptic membrane fragments prepared from Torpedo marmorata electric organ (specific activity,>4μmol of Naja nigricollis α[³H]toxin per mg of protein) exhibit, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, two major protein bands of apparent molecular weight 40,000 and 43,000, respectively. Dissolution of these membranes by the nondenaturing detergents Triton X 100 and Berol 043 followed by standard fractionation yielded (i) the 9S acetylcholine receptor protein which still binds the α-[³H]toxin and after further purification yielded, in the presence of sodium dodecyl sulfate, the 40,000 dalton component, covalently labeled by the affinity reagent 4 (N maleimido)phenyl[³H]trimethylammonium; only serine was found as the NH₂ terminal amino acid of this protein; and (ii) a high molecular weight aggregate named 43,000 protein which was resolved in denaturing gels almost exclusively as the 43,000 dalton band. In the absence of detergents, the 43,000 protein binds compounds known to interact with the acetylcholine ionophore: a fluorescent local anesthetic quinacrine and histrionicotoxin (apparent dissociation constant, 7 ± 1 x 10^-7 M). The regulation of quinacrine fluorescence by carbamylcholine, observed in the intact membrane, no longer occurs with the isolated 43,000 component.