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

Figures

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  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).

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  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.

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  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.

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  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).

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  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.

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  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.

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  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).

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  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.