Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor: The Role of Glutamate Residues 97, 224,and 235
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The binding of the fluorescein-labeled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino- (N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilized ligand-gated ion channel, the type- ...
The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstit ...
We have expressed the 5-HT3 receptor with two transient expression sytems: the Semliki Forest virus and the HEK293EBNA. Both systems allow the rapid expression of functional receptor protein at very high level: up to 107 receptors per cell. Large scale pro ...
The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while ...
A fluorescently labeled ligand for the 5-HT3 serotonin receptor was synthesized and its sub-nanomolar affinity for the purified, detergent solubilized receptor was measured. The change in the ligand's fluorescence upon receptor binding was used to directly ...
The thermodn. and kinetics of ligand binding to the purified serotonin 5HT3 receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3,9-tetrahydro-3-[(5-methyl-1H ...
Novel fluorescence approaches to investigate ligand recognition and structure of G protein-coupled receptors in native membranes have been developed. These methods combine the biosynthetic incorporation of unnatural fluorescent amino acids at known sites i ...
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 ves ...
A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed ...
Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system. The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcri ...