Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of α1 glycine receptors in HEK293 cells

• Published in: Journal of neuroscience methods 2002, Vol.113 (2), p.207-214
• Main Authors: Groot-Kormelink, Paul J, Beato, Marco, Finotti, Chiara, Harvey, Robert J, Sivilotti, Lucia G
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Glycine receptor; Ligand-gated ion channels; Plasmid ratio approach; Single-channel recording; Transfection; Vertebrates: nervous system and sense organs; α1 subunit
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/S0165-0270(01)00500-3

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 α1 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 α1-containing plasmid and empty plasmid vector. The maximum amplitude of glycine-evoked currents was reliably dependent on the percentage of α1-containing plasmid. Optimum results for steady-state single channel experiments at low glycine concentrations were obtained with 5% of α1 plasmid DNA in the transfection mix.