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

A fluorescently labelled ligand for the 5-HT3 serotonin receptor was synthe sised and its sub-nanomolar affinity for the purified, detergent solubilise d receptor was measured. The change in the ligand's fluorescence upon recep tor binding was used to directly measure its dissociation constant for rece ptor 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 s uitable for high-through-put screening. Therefore, the receptor was immobil ised via its C-terminal His-tag onto a nitrilotriacetic acid-modified quart z surface. The affinities of both the fluorescent ligand and several non-fl uorescent compounds were rapidly determined by the TIRF assay, and were sho wn to agree well with both the solution and classical radioligand binding a ssays. This indicated that the functional integrity of the receptor was pre served at the sensor surface. Due to the extreme sensitivity of the TIRF as say allows to obtain a complete pharmacological affinity profile of a quant ity of receptor provided by a small number of highly-expressing cells.