Interaction of nicotinic acetylcholine receptor with two monoclonal antibodies recognizing different epitopes.
The interactions of nicotinic acetylcholine receptor (nAChR) with two monoclonal antibodies (mAb370A and mAb371A) which block the agonist-induced ion flux into nicotinic acetylcholine receptor vesicles [Donnelly, D., Mihovilovic, M., Gonzalez-Ros, J. M., Ferragut, J. A., Richman, D., & Martinez-Carrion, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7999] have been studied by a combination of immunochemical and spectroscopic techniques. Both mAbs are specific for the alpha-subunit of the receptor, but they recognize different epitopes. We have detected specific binding of the mAb370A to a synthetic peptide corresponding to residues alpha 187-205, a sequence known to contain the alpha-bungarotoxin binding site. By contrast, mAb371A seems to recognize an epitope which is largely silent after proteolytic digestion of the subunit. Binding of mAb370A to the receptor is inhibited by cholinergic agonist and alpha-neurotoxins but not by competitive antagonists or local anesthetics. By contrast, none of these ligands interferes with binding of mAb371A. The spectroscopic properties of the fluorescent probe ethidium have been used to investigate the effect of the mAbs on the interaction of the agonist carbamylcholine with nAChR in membranes. mAb370A, but not mAb371A, blocks both the agonist-induced increase in the fluorescence intensity of receptor-bound ethidium and the agonist-induced increase in the polarization value of the probe. In addition, measurements of ethidium binding followed by stopped-flow techniques showed that mAb370A, but not mAb371A, blocked the agonist-induced association of the probe to nAChR membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Chinchetru
M A
MA
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110.
Marquez
J
J
Garcia-Borron
J C
JC
Richman
D P
DP
Martinez-Carrion
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D000939:Epitopes; D004996:Ethidium / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000276:immunology* / Q000378:metabolism; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D011978:Receptors, Nicotinic; D004996:Ethidium