Expression of the human 5-HT3A receptor using the baculovirus- and Epstein-Barr virus (EBV)-based expression systems

Date

2000-08

Journal Title

Journal ISSN

Volume Title

Publisher

Texas Tech University

Abstract

Due to the scarcity of 5-HT3A receptors in native tissue, it is extremely difficult to isolate sufficient quantities of protein to perform structural and biophysical studies on them. High-level heterologous expression systems provide a means of overcoming this limitation. The cDNA ofthe human 5-HT3A receptor with a hexa-hisfidine at its Cterminus was constructed with a PCR cloning strategy and was confirmed with dideoxy sequencing. The function ofthe recombinant 5-HT3A receptor was examined using the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiological recordings. Application of serotonin to recombinant receptors elicited inward sodium currents. No significant difference was revealed in the EC50S of serotonin in wild-type and 5-HT3A-His6 receptors. The cDNA for the human 5-HT3AHis6 receptor was subcloned into a baculovirus transfer plasmid and expressed in baculovirus-infected Sf9 insect cells. The expression ofthe human 5-HT3A-His6 receptor was assessed with both Western blot analysis and nickel affinity purification. In addition, saturation-binding experiments were carried out with crude membranes from baculovirus-infected insect cells and using affinity-purified receptors with the 5-HT3R antagonist [^H] GR65630. The K<i value for 5-HT3A-His6 receptors was 0.6 nM. Finally, the expression ofthe human 5-HT3A-His6 cDNA in 293EBNA (a human HEK293 cell line stably-transfected with the Epstein Barr virus nuclear antigen gene) cells has also been explored.

Description

Keywords

Serotonin, Epstein-Barr virus, Molecular cloning, Baculoviruses, Serotonin, Ligand binding (Biochemistry)

Citation