Antibody-induced conformational changes in the Torpedo nicotinic acetylcholine receptor: a fluorescence study.
Quantitative fluorescence spectroscopy was used to develop a structural picture of the effects of two monoclonal antibodies (mAbs) on the conformation of the Torpedo nicotinic acetylcholine receptor (nAChR). The two mAbs (A6 and B1) examined selectively blocked ligand binding to either the high-affinity (A) or the low-affinity (B) binding sites for agonists/competitive antagonists. The distances between dansyl-C6-choline bound to the unblocked agonist/competitive antagonist binding site and one of two lipophilic probes (C12-eosin or C18-rhodamine) partitioned into the lipid membrane were estimated by using fluorescence resonance energy transfer. Control experiments demonstrated that both mAbs decreased the affinity and fluorescence lifetime of receptor-bound dansyl-C6-choline. The binding of the B1 mAb to the B site did not significantly change the calculated distance between the unblocked A binding site and the membrane surface. However, the binding of the A6 mAb to the A site induced the B site to move into close proximity to the lipid membrane. This conformational change was confirmed by a 45-fold increase in the paramagnetic quenching of the B-site-bound dansyl-C6-choline fluorescence by lipid-intercalated 5-doxylstearate. The results indicate that these mAbs not only selectively block ligand binding to either the A or the B acetylcholine sites but also, in the case of the A6 mAb, induce global conformational changes of the receptor, which appear to involve a movement of the B binding site into close proximity of the lipid membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Valenzuela
C F
CF
Division of Biomedical Sciences, University of California, Riverside 92521-0121.
Dowding
A J
AJ
Arias
H R
HR
Johnson
D A
DA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites / Q000276:immunology; D002794:Choline / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000276:immunology; D013050:Spectrometry, Fluorescence; D014101:Torpedo
D000911:Antibodies, Monoclonal; D003619:Dansyl Compounds; D011978:Receptors, Nicotinic; D002794:Choline