Expression of ligand-gated ion channels with the Semliki forest virus expression system

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system. The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P-2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P-2x virus stocks. Infection of BHK, CHO and RTN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 x 10(6) 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P-2x receptor per mg protein. Functional responses to the P-2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by Ca-45(2+) influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P-2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT(3)His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermenter culture with SFV-5-HT(3)His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 x 10(6) receptors per cell.