Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tether ed membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural int egrity of the surface-reconstituted receptor was probed using monoclonal an tibodies, demonstrating that approximately 65% of the receptors present the ir ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the re ceptor in tethered membranes was investigated with Fourier transform infrar ed spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed a gainst the ligand-binding site which yielded dissociation constants in agre ement with radioligand binding assays. The presented method for the functio nal immobilisation of the nicotinic acetylcholine receptor in tethered memb ranes might be generally applicable to other membrane proteins. (C) 2000 El sevier Science B.V. All rights reserved.