High throughput clone screening on overexpressed hERG1 and Kv1.3 potassium channels using ion channel reader (ICR) label free technology

Abstract

Pharmacological studies aimed at the development of newly synthesized drugs directed against ion channels (as well as genetic studies of ion channel mutations) involve the development and use of transfected cells. However, the identification of the best clone, in terms of transfection efficiency, is often a time consuming procedure when performed through traditional methods such as manual patch-clamp. On the other hand, the use of other faster techniques, such as for example the IF, are not informative on the effective biological functionality of the transfected ion channel(s). In the present work, we used the high throughput automated ion channel reader (ICR) technology (ICR8000 Aurora Biomed Inc.) that combine atomic absorption spectroscopy with a patented microsampling process to accurately measure ion flux in cell-based screening assays. This technology indeed helped us to evaluate the transfection efficiency of hERG1 and hKv1.3 channels respectively on the HEK-293 and CHO cellular models. Moreover, as proof of the validity of this innovative method, we have corroborated these data with the functional characterization of the potassium currents carried out by the same clones through patch-clamp recordings. The results obtained in our study are promising and represent a valid methodological strategy to screen a large number of clones simultaneously and to pharmacologically evaluate their functionality within an extremely faster timeframe.

Keywords: ICR8000; Potassium channels; Rubidium efflux; hERG1; hKv1.3.

Fig. 1

CHO-transfected clones screening. Comparison of the rubidium effluxes between A) the 24 CHO-hERG1 clones and B) the 24 CHO-Kv1.3 clones object of this study. The efflux values are normalized to the efflux measured in the CHO-wt (here shown as a dashed line).

Fig. 2

CHO-transfected clones pharmacological test. Bar graph showing the effects of A) E4031 and B) PSORA-4 at the concentration of 10 μM on the two of the best clones among CHO-hERG1 and CHO-Kv1.3 cells performed with the rubidium efflux assay. Values are normalized to the control condition and here reported as a percentage. For statistical significance, two-tail unpaired T-test was used. *p < 0.0332; **p < 0.0021, ***p < 0.0002 and ****p < 0.0001.

Fig. 3

Transfected clones high-throughput dose-response curves. A) E4031 on HEK-hERG1 transfected cells (CL5, IC₅₀ = 0.399 μM) and B) on CHO-hERG1 transfected cells (CL6, IC₅₀ = 0.985 μM). C) PSORA-4 on CHO-Kv1.3 transfected cells (CL14, IC₅₀ = 0.08 μM). Values are normalized to the control condition.

Fig. 4

Representative traces of outward potassium currents evoked in A) HEK-wt, B) CHO-wt and C) CHO-hERG1 C6 clones (red: overprinted average traces). The insets represent the pulse protocols used for the electrophysiological recordings. D-F) Representative traces of both outward and inward potassium currents on the same clones. G-I) Representative traces of outward potassium currents evoked in A) HEK-wt, B) CHO-wt and C) CHO-Kv1.3 C6 clones (red: overprinted average traces).

Fig. 5

A-C) Representative outward and tail potassium current recordings measured in hERG1-transfected clones (6, 13 and 17) in control conditions. D-F) Representative outward and tail potassium current recordings measured in the same clones (6, 13 and 17) in the presence of 1 μM E4031.

Fig. 6

A-B) Average outward and tail potassium currents measured in each clone in control conditions. For statistical significance, two-tail unpaired T-test was used. *p < 0.0332; **p < 0.0021, ***p < 0.0002 and ****p < 0.0001. C-D) Average outward and tail potassium currents measured in each clone in the presence of 1 μM E4031.

Fig. 7

Electrophysiological properties of hERG1 current. A-C) I–V plots of outward hERG1 currents measured in hERG1-transfected clones (respectively 6, 13 and 17) in control conditions (black) and in the presence of 1 μM E4031 (red). D-F) I–V plots of tail hERG1 currents measured in the same clones in control conditions (black) and in the presence of 1 μM E4031 (red).

Fig. 8

Representative traces of CHO-Kv1.3 clones A) clone 6, B) clone 14, C) clone 17 and D) clone 24 recorded in control conditions (black) and in the presence of 1 μM PSORA-4 (blue). Pulse protocols used are the same shown in Fig. 4G. E) Average outward potassium currents measured in each clone in control conditions and F) in the presence of 1 μM PSORA-4.