title: Expression of ligand-gated ion channels with the Semliki Forest virus expression system.
abstract: 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 P2x 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-P2x virus stocks. Infection of BHK, CHO and RIN 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 P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x 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-HT3His 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 fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 x 10(6) receptors per cell.
authors.affiliation: Glaxo Wellcome, Medicines Research Centre, Stevenage, Herts, UK.
authors.forename: K
authors.initials: K
authors.lastname: Lundstrom
authors.forename: A
authors.initials: A
authors.lastname: Michel
authors.forename: H
authors.initials: H
authors.lastname: Blasey
authors.forename: A R
authors.initials: AR
authors.lastname: Bernard
authors.forename: R
authors.initials: R
authors.lastname: Hovius
authors.forename: H
authors.initials: H
authors.lastname: Vogel
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D006224:Cricetinae; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3; D012672:Semliki forest virus
chemical_list: D015220:Calcium Channels; D018048:Receptors, Purinergic P2; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D002118:Calcium