Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines

Abstract

Background: Many molecular studies of ion channel function rely on the ability to obtain high quality voltage clamp recordings using the patch clamp technique. For a variety of channel types studied in mammalian cell heterologous expression systems, the lack of experimenter control over expression levels severely hinders the ability to obtain a high percentage of cells with an expression level suitable for high quality recordings. Moreover, it has been nearly impossible to obtain expression levels in mammalian cells well suited for single channel recordings. We describe here the use of an inducible promoter system in a stably transfected mammalian cell line that produces nearly 100% success in obtaining ion channel expression levels suitable for either whole cell or single ion channel studies.

Control of Single Channel Conductance in the Outer Vestibule of the Kv2.1 Potassium Channel

“…In the experiments of Fig. 3 , as well as other types of experiments relevant to this study, channels expressed in HEK and CHO cells produced identical results (see Trapani and Korn, 2003 ).…”

“…We created a simple model based on the hypothesis that [K + ]-dependent changes in current magnitude could be accounted for entirely by changes in single channel conductance. This was well grounded in the experimental observation that the presence of the lysine at position 356 reduced conductance ( Consiglio et al, 2003 ; Trapani and Korn, 2003 ). We then used macroscopic and single channel conductance measurements to determine the large conductance magnitude, and consequently predict the small conductance magnitude and percent of channels in each conductance state, that would produce the observed [K + ]-dependent change in macroscopic current.…”

“…Wild-type Kv2.1, a Kv2.1 channel in which two outer vestibule lysines were neutralized (Kv2.1 K356G K382V), and a Kv2.1 channel in which one of the two outer vestibule lysines was neutralized (Kv2.1 K356G), were studied in stably transfected CHO cells. For these experiments, channel expression was under the control of a tetracycline (Tet)-inducible promoter, so that expression levels could be controlled (see Trapani and Korn, 2003 ). For whole cell recordings, channel expression was initiated by incubating cells in 1 μg/ml tetracycline for 2 h on the day of recording.…”

“…This was accomplished by using a CHO cell line in which channel protein expression was under the control of a Tet-inducible promoter. When cells were maintained in media made with Tet-free FBS, channel expression was reduced to a level that was suitable for single channel recording ( Trapani and Korn, 2003 ). Moreover, endogenous K + channel expression is so low in CHO cells that recordings are not detectably contaminated by endogenous currents ( Trapani and Korn, 2003 ).…”

Optical measurement of voltage sensing by endogenous ion channels

“…Kv2.1 construct (Kv2.1-CHO) (Trapani & Korn 2003), were cultured as described previously 7 (Tilley et al 2014). To induce channel expression in Kv2.1-TREx-CHO cells, 1 µg/mL 8 minocycline (Enzo Life Sciences, Catalog # ALX-380-109-M050), prepared in 70% ethanol at 2 9 mg/mL, was added to the maintenance media to induce K + expression.…”