ACETYLCHOLINE AND LOCAL-ANESTHETIC BINDING TO TORPEDO NICOTINIC POSTSYNAPTIC MEMBRANES AFTER REMOVAL OF NON-RECEPTOR PEPTIDES

After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent M(r) 41.000, 50.000, and 65.000 (i.e. the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of M(r) 43.000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of dimethisoquin were quantitatively unaltered. In the presence of 30 μM carbamoylcholine to occupy acetylcholine binding sites [14C]-meproadifen [2-(diethylmethylaminoethyl)-2-2-diphenylvalerate iodide] was bound with a dissociation constant, K[D] of 0.3 ± 0.1 μM to 0.3 ± 0.1 site per [3H]α-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of M(r) 43.000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.