Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of alpha 1 glycine receptors in HEK293 cells.
In single-channel recording, optimal yield of kinetic data is achieved if simultaneous activations of more than one channel are few. When recordings are obtained from recombinant channels, it is therefore important to control the level of expression of the channel at the cell surface, while maintaining a high efficiency of transfection. In the present study, we optimised transfection protocols for single-channel recording from recombinant rat alpha 1 glycine receptors expressed in HEK293 cells. High transfection efficiency was achieved with lipofection (up to 70%). Lipofected cells however did not lend themselves to excised patch recording because of seal instability, especially obvious at hyperpolarised holding potentials. High quality excised patch recordings were reliably achieved with the calcium phosphate-DNA coprecipitation method, with transfection efficiencies around 40%. We achieved good control of the level of receptor expression by a plasmid ratio approach which kept the total amount of plasmid transfected constant while varying the ratio between alpha 1-containing plasmid and empty plasmid vector. The maximum amplitude of glycine-evoked currents was reliably dependent on the percentage of alpha 1-containing plasmid. Optimum results for steady-state single channel experiments at low glycine concentrations were obtained with 5% of alpha 1 plasmid DNA in the transfection mix.
Groot-Kormelink
Paul J
PJ
Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, WC1N 1AX London, UK.
Beato
Marco
M
Finotti
Chiara
C
Harvey
Robert J
RJ
Sivilotti
Lucia G
LG
D002130:Calcium Phosphates; D002478:Cells, Cultured; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D018408:Patch-Clamp Techniques / Q000379:methods* / Q000592:standards; D010957:Plasmids / Q000235:genetics*; D018009:Receptors, Glycine / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods* / Q000592:standards
D002130:Calcium Phosphates; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018009:Receptors, Glycine; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous