A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

Abstract

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K(+) channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

Keywords: electrophysiology; fluorescent imaging; heterologous expression; human embryonic kidney 293 cells; membrane proteins; transfection.

Figure 1

Transfection and expression efficiencies of AtGORK tagged with Lumio™and AtGORK fused with EmGFP in HEK-293 cells. (A) HEK-293 cells transfected with AtGORK-cLumio™ expression vector has reduced transfection efficiency as compared to the expression of the positive control p64 that is consistent across all three biological replicates. (B) Representative images showing the poor transfection efficiency of cells transfected with AtGORK-cLumio™ and the p64 positive control plasmids. (C) The expression of AtGORK in green fluorescence cells is lower than that of p64 using the Lumio™ system as determined by their pixel intensities. (D) The AtGORK-EmGFP has a higher transfection efficiency and protein expression than that of the nLumio™-tagged p64 in HEK-293 cells. A typical 20 × magnification of 293FT cell image contains 2560 × 1920 pixels and is converted to an 8-bit byte image based on a scale of 0–255 using ImageJ (Schneider et al., 2012). The lower limit cut-off representing appreciable and above background fluorescence, was set at 10 pixels. Transfection efficiency and protein expression of more than 100 cells in each viewing field were analyzed (see Section Procedure for analysis).

Figure 2

Whole-cell current-voltage measurements of AtGORK-EmGFP in 293FT cells. (A) Bright field and fluorescence images of (i) 293FT and (ii) AtGORK-EmGFP—expressing 293FT single cell at 20 × magnification field view (scale bar = 20 μm). 293FT cells were transfected with 2.5 μg of Vivid Colors™ pcDNA™6.2/EmGFP-DEST Gateway^® expression vector containing the AtGORK insert using Lipofectamine^® 3000 in a “reverse” format (see Section Procedure for description). (B) Voltage- and time-dependent (i) and I-V plot (ii) properties of the intrinsic K⁺ channels in HEK-293 cells in response to a series of depolarizing square pulse (from a holding potential, H_V = −52 mV in 20 mV increments). Inset: A current showing the fast activation and relatively slower inactivation kinetics of intrinsic K⁺ currents upon depolarization. (C) Whole-cell patch-clamp recordings (i) and I-V plot (ii) of intrinsic K⁺ and AtGORK channels currents, starting from H_V = −52 mV in 20 mV increments in AtGORK-EmGFP—expressing 293FT single cell. Inset: A representative current demonstrating the slowly activating outward currents of AtGORK upon depolarization.

Figure 3

Comparison of different transfection reagents on the transfection and expression efficiencies of AtGORK-EmGFP in HEK-293 cells. HEK-293 cells transfected with AtGORK-EmGFP were used to examine the performance of different transfection reagents in a “reverse” format (see Section Procedure for definition). In general, Lipofectamine^® 3000 outperforms Lipofectamine^® 2000 and FuGENE^® HD in both transfection efficiency (A,B) and protein expression (C). Lipofectamine^® 2000 and FuGENE^® HD have comparable transfection efficiencies but lower than that of Lipofectamine^® 3000. FuGENE^® HD seems to yield protein expression levels comparable to that of Lipofectamine 3000. A typical 20 × HEK cell image contains 2560 × 1920 pixels and is converted to an 8-bit byte image that on a scale of 0–255 using ImageJ. The lower limit cut-off that represents appreciable and above background fluorescence, was set at 10 pixels for biological replicate 1 but 40 pixels for replicates 2 and 3. Transfection efficiency and protein expression of more than 200 cells in each viewing area were analyzed (see Section Procedure for analysis).

Figure 4

Effect of different transfection methods on the transfection and expression efficiencies of AtGORK-EmGFP in HEK-293 cells. HEK-293 cells transfected with AtGORK-EmGFP were used to examine the performance of different transfection format: “standard,” “reverse,” and “double” (see Section Procedure for definition) using the Lipofectamine^® 3000 reagent. The “standard” transfection method outperforms both the “reverse” and “double” transfection format respectively, in 2 out of 3 biological repeats in terms of transfection efficiency (A,B). The “standard” transfection protocol yields higher protein expression levels in only 1 biological replicate but yield comparable expression levels to the other transfection methods in replicates 2 and 3 (C). A typical 20 × HEK cell image contains 2560 × 1920 pixels and is converted to an 8-bit byte image that is on a scale of 0–255 using ImageJ. The lower limit cut-off representing appreciable and above background fluorescence, was set at 10 pixels for 40 pixels. Transfection efficiency and protein expression of more than 100 cells in each viewing field were analyzed (see Section Procedure for analysis).