Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides.
After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent Mr 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 Mr 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 30 micron carbamoylcholine to occupy acetylcholine binding sites, [14C]-meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide ] was bound with a dissociation constant, KD, of 0.3 +/- 0.1 micron to 0.3 +/- 0.1 site per [3H]alpha-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 Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.
D000109:Acetylcholine / Q000378:metabolism*; D000779:Anesthetics, Local / Q000378:metabolism*; D000818:Animals; D002463:Cell Membrane Permeability; D004557:Electric Organ / Q000378:metabolism; D005399:Fishes; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D007546:Isoquinolines / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D011335:Proadifen / Q000031:analogs & derivatives* / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism*
D000779:Anesthetics, Local; D007473:Ion Channels; D007546:Isoquinolines; D008565:Membrane Proteins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; C019001:meproadifen; D012964:Sodium; D011335:Proadifen; D000109:Acetylcholine