Interaction of nicotinic acetylcholine receptor with two monoclonal antibodies recognizing different epitopes

Miguel A. Chinchetru, Javier Marquez, Jose C. Garcia-Borron, David P Richman, Marino Martinez-Carrion

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14 Scopus citations


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 α-subunit of the receptor, but they recognize different epitopes. We have detected specific binding of the mAb370A to a synthetic peptide corresponding to residues α187-205, a sequence known to contain the α-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 agonists and α-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. Therefore, mAb370A acts as a specific nAChR ligand which binds to an epitope which contains parts of the α-subunit sequence between residues 187-205 and competes with cholinergic ligands blocking the receptor ion channel opening response. By contrast, mAb371A acts through an, as yet, uncharacterized long-distance, non-competitive mechanism, the latter being consistent with a conformational change of the nAChR induced by mAb371A, as reported for interactions of neuraminidase and antibodies [Colman, P. M., Laver, W. G., Varghese, J. N., Baker, A. T., Tulloch, P. A., Air, G. M., & Webster, R. G. (1987) Nature 326, 358-363]. Such conformational changes could shift the close-open channel equilibrium toward a closed conformation or can prevent protein movements necessary for ion channel opening.

Original languageEnglish (US)
Pages (from-to)4222-4229
Number of pages8
Issue number10
StatePublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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