AFFINITY LABELING OF NEURONAL ACETYLCHOLINE-RECEPTOR SUBUNITS WITH AN ALPHA-NEUROTOXIN THAT BLOCKS RECEPTOR FUNCTION

  • 1 August 1987
    • journal article
    • research article
    • Vol. 7  (8) , 2547-2555
Abstract
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. Coexistence of Bgt 3.1 and mAb 35 binding sites was demonstrated in a similar manner for each of the several potential AChR subpopulations inferred previously from biphasic dissociation kinetics of bound Bgt 3.1 on the neurons and from incomplete modulation of mAb 35 binding sites on the neurons by Bgt 3.1. Gel electrophoresis of the immune-precipitated, photoaffinity-labeled AChR under denaturing conditions revealed a specifically labeled band ith a Mr of 66,000. Subtracting 7000 for the size of the covalently attached toxin yields a Mr of 59,000 for the corresponding receptor subunit likely to be associated with the active site. On some occasions, a second labeled band of 61,000 was observed, corresponding to a receptor polypeptide of 54,000. No evidence was obtained for a population of AChRs that selectively bound either Bgt 3.1 or mAb 35.

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