title: A novel and efficient method for the stable expression of heteromeric ion channels in mammalian cells.
abstract: The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.
authors.affiliation: Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Switzerland.
authors.forename: E
authors.initials: E
authors.lastname: Kawashima
authors.forename: D
authors.initials: D
authors.lastname: Estoppey
authors.forename: C
authors.initials: C
authors.lastname: Virginio
authors.forename: D
authors.initials: D
authors.lastname: Fahmi
authors.forename: S
authors.initials: S
authors.lastname: Rees
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
authors.forename: R A
authors.initials: RA
authors.lastname: North
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004686:Encephalomyocarditis virus; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics*; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D015220:Calcium Channels; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate