A novel and efficient method for the stable expression of heteromeric ion channels in mammalian cells.
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.
Kawashima
E
E
Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Switzerland.
Estoppey
D
D
Virginio
C
C
Fahmi
D
D
Rees
S
S
Surprenant
A
A
North
R A
RA
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
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