A desensitized form of neuronal acetylcholine receptor detected by 3H-nicotine binding on bovine adrenal chromaffin cells.
A nicotinic acetylcholine receptor (AChR) on bovine adrenal chromaffin cells in culture has previously been identified using the alpha-neurotoxin n-Bgt and the monoclonal antibody mAb 35. Here, we report that the cells have 2 classes of high-affinity binding sites for 3H-nicotine, one being associated with the AChR and the other being associated with the alpha-bungarotoxin binding component that is distinct from the AChR. Scatchard analysis of 3H-nicotine binding to the AChR site yields a KD of 20 +/- 3 nM and a Bmax of 104 +/- 12 fmol/mg protein. Nicotinic antagonists block 3H-nicotine binding to the AChR site with the same rank order of potency and affinity with which they block nicotine-induced catecholamine release from the cells. About 80% of the AChRs are on the cell surface, as judged by the distribution of both 3H-nicotine binding and 125I-mAb 35 binding to the receptor, and the ratio of nicotine/mAb 35 binding to the AChR on the cell surface is approximately 1:1. Chronic treatment of the cells with mAb 35 results in receptor modulation such that all of AChR-related nicotine binding is lost from the cell surface, and all of the functional response to nicotine is lost as well. The results confirm that 3H-nicotine binding is associated with AChRs on the cells. The 3H-nicotine binding observed to the AChR represents binding to a desensitized form of the receptor having increased affinity for agonists and unchanged affinity for antagonists. This conclusion derives from the following observations. The KiS for agonist competition of 3H-nicotine binding indicate agonist affinities several orders of magnitude greater than do the KDS measured for receptor activation. Exposing cultures to low concentrations of nicotine and substance P causes receptor densensitization, and the concentration dependence of the nicotine-induced desensitization displays an EC50 of 20 nM, in good agreement with the KD obtained from equilibrium binding studies with 3H-nicotine. In addition, the rate of 3H-nicotine binding is increased both by substance P, which enhances the rate of agonist-induced desensitization on adrenal chromaffin cells, and by preincubation with nicotine. The increased rate of association, together with the dissociation rate, yields a kinetically derived KD of 19 nM, again in good agreement with the KD obtained from equilibrium binding studies. These results demonstrate that the bovine adrenal chromaffin AChR is similar to AChRs from muscle and electric organ in undergoing an agonist-induced conversion to a desensitized state having increased affinity for agonists.
Higgins
L S
LS
Department of Biology, University of California, San Diego, La Jolla 92093.
Berg
D K
DK
D000311:Adrenal Glands / Q000166:cytology*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000648:ultrastructure*; D002838:Chromaffin System / Q000648:ultrastructure*; D007700:Kinetics; D009474:Neurons / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*
D011950:Receptors, Cholinergic; D009538:Nicotine