Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes.
Transient transfection of cultured mammalian cells is widely employed in the study of ionotropic glutamate receptors. Heteromeric expression is usually achieved by simultaneous transfection of various combinations of glutamate receptor subunit-encoding cDNAs. This approach is based on an "all-or-none" assumption, rarely verified experimentally, that any given cell expresses all subunits present during transfection. A similar assumption implicitly is made when cotransfection of a cDNA encoding a fluorescent marker protein is applied to distinguish transfected from untransfected cells. A further frequent assumption alleges that the ratio between cDNAs used in cotransfection experiments directs the assembly of receptor complexes in heterologous expression systems. To check the validity of these assumptions for ionotropic glutamate receptors as model transmembrane receptors, we generated fluorescently labeled receptor subunits and introduced them into HEK-293 cells by the calcium phosphate method. Analyzing the expression of multiple fusion proteins by confocal microscopy, we evaluated the coexpression efficiencies for various glutamate receptor cDNA combinations, cDNA amounts, and cDNA ratios. Several factors were found to influence the individual, cumulative, and cotransfection efficiencies, including the cDNA ratio, the nature of the expressed protein, and the specific combination of cotransfected cDNAs. After simultaneous transfection with equal amounts of several cDNAs, we demonstrate the consistent generation of several distinct populations of cells that express different receptor subunit combinations. The evidence we present suggests that cotransfected cells should always be independently tested for the expression of all target subunits before picking cells for the analysis of specific heteromeric receptor assemblies.
Ma
Zhan-Lu
ZL
Department of Biochemistry I-Receptor Biochemistry, Ruhr University Bochum, Bochum, Germany.
Werner
Markus
M
Körber
Christoph
C
Joshi
Illah
I
Hamad
Mohammad
M
Wahle
Petra
P
Hollmann
Michael
M
D002452:Cell Count / Q000379:methods; D002461:Cell Line, Transformed; D004558:Electric Stimulation / Q000379:methods; D015870:Gene Expression / Q000502:physiology*; D005819:Genetic Markers; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008164:Luminescent Proteins / Q000378:metabolism*; D008564:Membrane Potentials / Q000235:genetics; D018408:Patch-Clamp Techniques / Q000379:methods; D018092:Kainic Acid Receptors / Q000378:metabolism*; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods*; D000097806:GluK2 Kainate Receptor
D005819:Genetic Markers; D008164:Luminescent Proteins; D018092:Kainic Acid Receptors; D000097806:GluK2 Kainate Receptor