Affinity labeling of neuronal acetylcholine receptor subunits with an alpha-neurotoxin that blocks receptor function.
An alpha-neurotoxin, Bgt 3.1, has previously been shown to recognize the functional acetylcholine receptor (AChR) on chick autonomic neurons, since it specifically blocks receptor function and it binds to a class of sites on the neurons with the pharmacology, kinetics, and affinity expected for the receptor. A monoclonal antibody, mAb 35, to the main immunogenic region of muscle and electric organ AChR alpha subunit cross-reacts with a component on chick autonomic neurons that, from several lines of evidence, also appears to be the functional AChR. The identity of the antibody-binding component has remained in doubt, however, because previous studies indicated that in at least one instance a substantial discrepancy existed between the number of functional AChRs estimated physiologically and the number of putative AChRs detected by mAb 35 on the neurons. The present findings demonstrate that Bgt 3.1 and mAb 35 recognize the same AChRs on the neurons, and provide information about the stoichiometry of binding and the identity of subunits associated with active sites on the receptor. Chick ciliary ganglion neurons examined under a variety of growth and regulatory conditions in culture displayed a constant ratio of about 2:1 for mAb 35 and Bgt 3.1 binding to cell surface sites. Treatment of the cells with mAb 35 induced a substantial decrease in the number of Bgt 3.1 sites and vice versa. AChRs that had been covalently labeled with a photoaffinity derivative of 125I-Bgt 3.1 in situ and then solubilized were specifically immune-precipitated by mAb 35, demonstrating unequivocally that the receptors possessed both Bgt 3.1 and mAb 35 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D005726:Ganglia, Parasympathetic / Q000378:metabolism*; D008970:Molecular Weight; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D014403:Tubocurarine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D002217:Carbachol; D014403:Tubocurarine