Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System.

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT(3) receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1.

Localization and targeting of SCG10 to the trans-Golgi apparatus and growth cone vesicles.

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles.

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 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.

Ligand binding to the serotonin 5HT3 receptor studied with a novel fluorescent ligand.

The thermodynamics 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-imidazol-4-yl)methyl]-9-(3-amino-(N-fluo rescien-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiological investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissociation constant of 0.32 nM.