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

Josef G Trapani,Stephen J Korn

doi:10.1186/1471-2202-4-15

Abstract

BackgroundMany 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.ResultsWe used a tetracycline-regulated expression system to control K+ channel expression in a CHO (Chinese hamster ovary) cell line. Current magnitudes within a reasonably narrow range could be easily and reliably obtained for either macroscopic or single channel recordings. Macroscopic currents of 1 – 2 nA could be obtained in nearly 100% of cells tested. The desired expression level could be obtained within just 2 to 3 hours, and remained stable at room temperature. Very low expression levels of transfected channels could also be obtained, which resulted in a >70% success rate in the ability to record single channel currents from a patch. Moreover, at these low expression levels, it appeared that endogenous channels produced little or no contamination.ConclusionThis approach to controlling ion channel expression is relatively simple, greatly enhances the speed and efficiency with which high quality macroscopic current data can be collected, and makes it possible to easily and reliably record single channel currents in a mammalian cell heterologous expression system. Whereas we demonstrate the ability of this system to control expression levels of voltage-gated K+ channels, it should be applicable to all other channel types that express well in mammalian expression systems.

Highlights

Many molecular studies of ion channel function rely on the ability to obtain high quality voltage clamp recordings using the patch clamp technique
These studies usually utilize patch clamp techniques to examine the influence of experimental manipulations on the magnitude, gating kinetics and/or voltage-dependence of ionic currents passing through the channels
Potassium channel expression levels often result in currents in the 5 to 10 nA range at 0 mV

Summary

Introduction

Many molecular studies of ion channel function rely on the ability to obtain high quality voltage clamp recordings using the patch clamp technique. Potassium channel expression levels often result in currents in the 5 to 10 nA range at 0 mV This precludes quantitatively accurate voltage clamp recordings. The complete lack of control over expression in stable lines makes it impossible to obtain the range of expression levels required for different types of experiments. This lack of control, in both transient and stable transfection systems, significantly hinders experimental progress

Methods

Results

Conclusion