Investigation and Characterisation of Endogenous Potassium Voltage-Gated Ion Channel Currents in a Parental HEK Cell Line and Their Effect on an IonWorks Quattro hNaV1.5 Assay Using Transfected Cells

David Standing, Jacob Butterworth, David Downie, Andrew Powell. GlaxoSmithKline, Stevenage, UK

Frozen and fresh cell cultures of stably transfected HEK-293 cells are commonly used in drug screening assays for voltage gated sodium channel blockers. These assays routinely run on automated electrophysiology systems such as IonWorks and IonFlux.

Whilst they generally show a low level of native ion channel expression, HEK-293 cells have been shown to express endogenous voltage gated potassium channels in traditional patch clamp studies (Yu & Kerchner (1998), Bo Jiang et al (2002)). However there has been little or no published work on how these endogenous channels may be observed in automated systems such as IonWorks.

A selection of non-specific Na+ and K+ channel blockers were screened in an IonWorks Quattro PPC assay using a parental HEK cell line both before and after transfection with hNaV1.5. Both parental and transfected cells produced similar seal resistances, but a depolarisation to 0mV from -80mV produced mean inward currents of only 104(±183 standard deviation, n = 144)pA in the parental cells, which were 3% of the value of the transfected cells. No indication was found that endogenous ion channels interfered with the assay signal.

Further investigations were carried out on the parental cells using the IonWorks Quattro in HT mode. Appreciable outward currents were observed in this configuration (296(±360 standard deviation, n = 8)pA @ +40mV) which precisely matched those produced in patch clamp data using equivalent assay conditions by Yu & Kirchner (1998). These outward currents were characterised using the potassium channel blockers Amitryptiline, Tetraethylammonium and 4-aminotryptiline; these studies indicated that the currents showed a different profile to those published for supposedly identical HEK cells, and may be due to 6TM Shaker-related KV1.x channels (Alexander et al (2008)).

We therefore confirmed that HEK-293 cells are a suitable system for exogenous Na+ channel expression in IonWorks PPC assays, and suggest that the unexpected character of the endogenous currents observed (together with earlier profiling data) may indicate changes in the native expression characteristics of this long-maintained in-house cell line.

Yu SP, Kerchner GA (1998) “Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells”. J Neurosci Res 52(5):612-7

Bo Jiang, Xianfeng Sun, Kun Cao, Rui Wang (2002) “Endogenous KV channels in human embryonic kidney (HEK-293) cell”. Molecular & Cellular Biochemistry 238: 69-79

Alexander SPH, Mathie A, Peters JA (2008). Guide to Receptors and Channels (GRAC), 3rd edn. Br J Pharmacol 153 (Suppl. 2): S1–S209