Independent activation of the acetylcholine receptor from Torpedo californica at two sites.
Membrane vesicles enriched in acetylcholine receptor were prepared from the electroplax tissue of Torpedo californica. The receptor was reduced with dithiothreitol to expose a sulfhydryl group near the ligand binidng site and then treated in one of the following ways: (1) affinity alkylated treated in one of the following ways: (1) affinity alkylated with bromoacetylcholine, a receptor activator, (2) affinity alkylated with maleimidobenzyltrimethylammonium, a receptor inhibitor, or (3) reoxidized to the native state with dithiobis(2-nitrobenzoate). The affinity labels blocked half of the binding sites for alpha-bungarotoxin. The toxin sites not protected by the affinity labels were protected by carbamylcholine based on studies of toxin binding kinetics. The functional response of native and affinity-alkylated receptors was measured by a sodium ion flux procedure. In the absence of added cholinergic activators, only slow ion flux was observed. In the presence of carbamylcholine, a receptor activator, both native and modified membranes showed the increased sodium flux associated with functional receptors. The concentration of carbamylcholine required for a 50% maximal response was higher in the affinity-labeled membranes. Preincubation of the membranes with carbamylcholine blocked the increased ion flux, indicating that desensitization could be induced. The results provide evidence for the existence of two functional sites on the acetylcholine receptor. Each site corresponds to a bungarotoxin binding site and can be independently activated and desensitized.
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004557:Electric Organ / Q000378:metabolism*; D005399:Fishes; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*
D011950:Receptors, Cholinergic; D002217:Carbachol; D004228:Dithionitrobenzoic Acid; D000109:Acetylcholine