Ana-Paula Tairi

Characterization of a mouse serotonin 5-HT3 receptor purified from mammalian cells

Abstract: A serotonin 5‐HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one‐step purification yielding ∼50% of the histidine‐tagged 5‐HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild‐type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of ∼5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5‐HT3 receptor is a pentameric homooligomer. The secondary structure of the 5‐HT3 receptor as determined by circular dichroism appeared to consist of mainly α‐helices (50%) and β‐strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.

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 synthesised and its sub-nanomolar affinity for the purified, detergent solubilised receptor was measured. The change in the ligands 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.