ANTIBODY-INDUCED CONFORMATIONAL-CHANGES IN THE TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR - A FLUORESCENCE STUDY

Quantitative fluorescence spectroscopy was used to develop a structura l picture of the effects of two monoclonal antibodies (mAbs) on the co nformation of the Torpedo nicotinic acetylcholine receptor (nAChR). Th e 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-C-6-ch oline bound to the unblocked agonist/competitive antagonist binding si te and one of two lipophilic probes (C-12-eosin or C-18-rhodamine) par titioned 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-boun d dansyl-C-6-choline. The binding of the B1 mAb to the B site did not significantly change the calculated distance between the unblocked A b inding site and the membrane surface. However, the binding of the A6 m Ab to the A site induced the B site to move into close proximity to th e lipid membrane. This conformational change was confirmed by a 45-fol d increase in the paramagnetic quenching of the B-site-bound dansyl-C- 6-choline fluorescence by lipid-intercalated 5-doxylstearate. The resu lts 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, whi ch appear to involve a movement of the B binding site into close proxi mity of the lipid membrane. Because mAbs can induce substantial change s in the position of functional domains of the nAChR, mAbs appear to h ave the potential of dramatically altering epitope locations, and cons equently, conflicting results can potentially arise when mAbs are used to delineate structural details of the nAChR.