Ligand Binding to Nicotinic Acetylcholine Receptor Investigated by Surface Plasmon Resonance

Abstract

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A K(D) = 3.5 × 10(-)(6) M for the detergent-solubilized receptor and a K(D) = 1.4 × 10(-)(5) M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a K(D) = 4.5 × 10(-)(5) M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligand-receptor interactions in case of small ligand molecules by mass-sensitive techniques.

Sensitive detection of voltage transients using differential intensity surface plasmon resonance system

“…As they are sensitive to the optical properties of the dielectric medium, they have gained a great deal of interest in bio-medical research being used to probe molecular interactions within the evanescent field at the metal-dielectric interface [11], cell sensing [12] or imaging the cell membrane [13]. In addition to SPs sensitivity to the properties of the dielectric layer, they are also sensitive to the properties of the supporting metallic layer.…”

Self‐Assembled Microarrays of Attoliter Molecular Vessels

“…The presence of 10 % charged lipids (POPG) also increases the bending energy14 of the lipid membrane, which prevents vesicles from deforming and fusing upon immobilization. To establish a second barrier against intimate contact between surface and vesicles, we used lipids modified with a hydrophilic polymeric chain (PEG) 15. The PEG molecules induce a force that is entropic in nature and independent of the immobilization conditions (pH, ionic strength) that affect electrostatic‐based repulsion.…”

Receptor–ligand binding assays: Technologies and Applications

“…The sensitivity of the assay may be increased by immobilizing the ligand on the sensor surface thereby generating a large signal due to binding of the high-molecular mass receptor as described by Kröger et al [114] for the nicotinic acetylcholine receptor. In this approach, the biotinylated antagonist ␣-bungarotoxin was immobilized onto a streptavidin coated gold surface and the receptor was solubilized or reconstituted in liposomes in order to suppress non-specific binding.…”

“…carbachol and subsequently injected onto the biosensor. A high concentration of carbachol added to the receptor suspension gives rise to a small increase in the SPR response because of low receptor binding to the surface and vice versa [114].…”