Fluorescence techniques for fundamental and applied studies of membrane protein receptors: the 5-HT3 serotonin...

A fluorescently labelled ligand for the 5-HT3 serotonin receptor was synthesised and its sub-nanomolar affinity for the purified, detergent solubilised receptor was measured. The change in the ligand's fluorescence upon receptor binding was used to directly measure its dissociation constant for receptor binding, to determine the pharmacology of the receptor, and finally to characterise the binding site of the receptor. A total internal reflection fluorescence (TIRF) assay for the 5-HT3 receptor was developed, which is suitable for high-through-put screening. Therefore, the receptor was immobilised via its C-terminal His-tag onto a nitrilotriacetic acid-modified quartz surface. The affinities of both the fluorescent ligand and several non-fluorescent compounds were rapidly determined by the TIRF assay, and were shown to agree well with both the solution and classical radioligand binding assays. This indicated that the functional integrity of the receptor was preserved at the sensor surface. Due to the extreme sensitivity of the TIRF assay allows to obtain a complete pharmacological affinity profile of a quantity of receptor provided by a small number of highly-expressing cells.