Journal of Neuroscience, Vol 8, 1436-1446, Copyright © 1988 by Society for Neuroscience
A desensitized form of neuronal acetylcholine receptor detected by 3H- nicotine binding on bovine adrenal chromaffin cells [published erratum appears in J Neurosci 1988 Dec;8(12):preceding 4415]
LS Higgins and DK Berg
Department of Biology, University of California, San Diego, La Jolla 92093.
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.
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