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.

The Arabidopsis thaliana K+-Uptake Permease 5 (AtKUP5) Contains a Functional Cytosolic Adenylate Cyclase Essential for K+ Transport

“…Attempts were also made to see if HEK-293 cells expressing AtKUP5 show any novel/unusual currents at other depolarized voltages (from +120 to -80 mV in -20 mV decrements) but they did not (data not shown). Mostly, at these depolarized voltages and using our internal and external media (see section “Materials and Methods”), we only recorded the common intrinsic I A -like K + -current, arising from a channel native to HEK-293 cells ( Ooi et al, 2016 ).…”

Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method

“…This fact can explain the lower transfection rates obtained when lipofection was used. Moreover, the use of lipofection has been generally associated with transient transfection (Ooi et al, 2016;Fuge et al, 2017), a fact that limits its application in the production of transgenic animals (Bressan et al, 2008).…”

Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

“…We hypothesized that some factors involved in the GPLD1-induced modulation of the A␤ production are not sufficiently expressed in the H4 cell line. HEK293 cells have often been used to study APP processing and A␤ production (26) and also are known to express a wide range of membrane-bound and secreted proteins (27). Therefore, we transfected GPLD1 into HEK293 cells, collected the conditioned media, and added these HEK293-conditioned media to H4-APPsw cell cultures to investigate changes in A␤ production.…”