Application Note

Channel:hERG
Cells:
CHO®
Tools:
SyncroPatch 384PE

High Throughput Pharmacology of hERG
Channels on Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResultschannel

For the evaluation of the performance of hERG (CHO)

responsible for the repolarization of the IKr current incells, Seal Resistance, Cslow and the Series Resistance(Rseries) were determined from one experiment (Fig. 1) .
A 80

Abnormalities in this channel can lead to long or shortarrhythmias and anti-psychotics, it has become an20

Therefore, it is important to test new therapeutics foractions on the hERG channel early on in the drug
discovery process.
Here we present high quality data with reliable
pharmacology on hERG expressing CHO cells at ahigh throughput collected on the SyncroPatch® 384PE.
Current-voltage plots, and concentration response curves
for the compounds pimozide, astemizole, cisaprideand terfenadine are shown. The IC50 values for thesecompounds are within the expected range3-11 andsuccess rates of 80% for completed experiments wererecorded.

Download more Application Notes from www.nanion.de1040ΩG40Cells (%)

nal prokinetic agents (e.g. Cisapride), amongst others2.

Seal Resistance
C60

B

20200

<551
10 0-1
15 5-220 0-25>2
5include antihistamines (e.g. Terfenadine), gastrointesti-Cells (%)

QT syndrome by inhibiting the hERG channel. These0chemical structures have been reported to induce long>1ed
sa
bldiA large range of therapeutic agents with diverseΩ0

important target in compound safety screening.

Gchannel activity by certain compounds such as anti-40-1

maintaining cardiac function, and disturbances ofΩarrhythmia. Given the importance of this channel in60
Cells (%)

QT syndrome, leading to potentially fatal cardiac

start of experimentend of experiment

Cslow (pF)0<551
10 0-1
15 5-2
20 0-2
5>25potential.

ΩtheMactioninM

cardiacimportant

0

theis

50

ofchannel

potassium00

repolarization

This

a-5

cells1.

encodesΩ

cardiac

geneM

hERG

<100

TheRseries (MΩ)

Figure 1: Statistics of hERG expressing CHO cells recorded on one NPC384 chip on the SyncroPatch® 384PE
A Success rate (seal resistance) of individual CHO cells on theSyncroPatch® 384. Shown is a bar graph of seal resistances at the start(light blue) and end of the experiment (dark blue). B Bar graph of cell
capacitance (Cslow) and C Series Resistance (Rseries) values for CHO
cells expressing hERG.

Application Note
Currents mediated by hERG could be reliably recordedon the SyncroPatch® 384PE with a high success rate.
Figure 2 shows a screenshot of the data acquisition andanalysis software of the SyncroPatch® 384PE during anexperiment recording the current-voltage relationship ofhERG expressed in CHO cells. In this experiment, 84% ofcells had a seal resistance > 500 MΩ with a further 12%
with a seal resistance > 100 MΩ.

Figure 3: IV and inhibition of hERG channels expressed in CHO cells on theSyncroPatch® 384PE. A hERG-mediated current responses to a voltage step
protocol (see Fig. 2). B The corresponding current voltage relationship from(A) shows the typical IV curve of hERG channels. C Current response ofan exemplar cell elicited using a voltage step to -40 mV for 500 ms after adepolarizing step to 60 mV for 500 ms (holding potential -110 mV). The red
trace shows inhibition by 250 nM pimozide and the cursors and red boundingbox indicate the maximum peak amplitude used for plotting the online
analysis timeplot. D Corresponding online analysis showing peak amplitude
plotted against time. The online analysis is color-coded, the grey region
indicates the application of control solution including vehicle (0.3% DMSO)
and the blue region indicates application of 250nM pimozide.
Figure 2: Typical recording from hERG expressed in CHO cells on theSyncroPatch® 384PE. The screenshot shows the data acquisition and analysis
software used on the SyncroPatch® 384PE. Wells are color-coded based onseal resistance (green: Rmemb > 500 MΩ, blue: Rmemb = 100 - 500 MΩ, light
blue or grey: Rmemb < 100 MΩ or disabled). Shown are hERG-mediatedcurrents elicited using a voltage step protocol from -120 mV to 80 mV
increasing in 20 mV increments (after a depolarizing step to 60 mV). Theblack trace highlights the maximum current in response to a test potentialof -40 mV.

A summary of IC50 values and success rates for the 4compounds is shown in Table 1. The estimated IC50 values

agree well with those found in the literature3-11. The successrates for completed experiments were between 69 and82%.

Figure 3 shows the current-voltage relationship for an
exemplar hERG-expressing CHO cell and block of
the current by pimozide (250 nM). Figure 3B and3D show the online analysis for the current-voltage
relationshipandthepharmacology

experiment,

respectively. In Panel D, the vertical lines and colorcoded regions show addition and incubation ofcompound. The white region shows incubation incontrol solution (external wash solution), the grey region
incubation in vehicle (0.3% DMSO) and the blue region
incubation in 250 nM pimozide. Each cell was exposedtoasingleconcentration
thewholeconcentration
responseplate.

ofcurve

Thepimozide

andcalculated

averagetheacrossconcentration

response curves for four hERG-active compounds areshown in Figure 4. Only cells which

satisfied certain

quality control criteria (Rmemb, Rseries, Cm and peakFigure 4: Average concentration response curves for 4 different hERG
blockers on the SyncroPatch® 384PE. The concentration response curves wereconstructed across the whole plate as shown in Figure 5. The SyncroPatch®
384PE analysis software (DataControl® 384) was used to calculate theaverage concentration response curves, normalized to maximum blockand fitted with a standard Hill-equation. A summary of the IC50 values andsuccess rates is shown in Table 1.

current amplitude) were included in the analysis.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch® 384PE. Screenshot of depiction of online analysis
data of hERG expressing CHO cells as recorded on one NPC-384 patch clamp chip. Three hundred and eighty-four small color-coded pictures as seen in
the upper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 500 MΩ), blue (Rmemb = 100 – 500 MΩ), light
blue or grey (Rmemb < 100 MΩ or cells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on theright. Graphs show current amplitudes of hERG channels during the voltage step to -40mV during application of control solution (white region), vehicle(0.3% DMSO; grey region) and inhibition by pimozide (blue region).

Exp end and chip
unload10 - 12 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash + 1 vehicle wash +1 compound addition

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch® 384PE. The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) fora single point concentration response curve on hERG-mediated currents took approximately 17-19 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note
CompoundIC50 (nM)

Successrate (%)

Literaturerange (nM)Pimozide

5.9 (301)

78

1 - 183,4

Astemizole

19.8 (315)82

0.9 - 265,6,7

Cisapride54.6 (292)76

6.5 - 443,4,6,8

Terfenadine

60.8 (264)69

7 - 2043,6,9,10,11

this example, white shows incubation in control solution,
grey is vehicle (0.3% DMSO) and blue is pimozide.
In conclusion, hERG expressed in CHO cells canbe recorded on the SyncroPatch® 384PE with highsuccess rates for completed experiments (typically>70%). The timeline of each experiment was about 17-19

Table 1: IC50 values for pimozide, astemizole, cisapride and terfenadineon hERG-mediated currents recorded on the SyncroPatch® 384PE. Shown
are IC50 values (number of cells shown in brackets), success rate forcompleted experiments and the expected literature IC50 values. All IC50
values recorded on the SyncroPatch® 384PE agree well with the literature
values3-11.

Figure 5 shows a screenshot of the SyncroPatch® 384
software during an experiment. A color-coded overview (based on seal resistance in this case) of all 384
wells gives the user a good impression of the successrate of the experiment. The user can choose whether tovisualize raw traces or online analysis. Here, online analysis
is chosen and the graphs represent current amplitude
versustime.

Anindividual

wellcanbehighlighted to monitor progression of the experiment.
In the Online Analysis view, the time points at which
solution additions have been made are indicated byvertical lines, as well as different background colors. In

References

minutes (start – end) and included wash with control
solution, wash with vehicle and > 5 min incubationin 1 concentration of blocker. The current-voltage
relationship of hERG recorded on the SyncroPatch®
384PE is in good agreement with the literature1. The IC50’s

calculated using the SyncroPatch® 384PE’s analysis

software, DataControl® 384, of pimozide, astemizole,cisapride and terfenadine were in good agreement withthose found in the literature3-11.
The SyncroPatch® 384PE is a high throughput and highly
reliable automated patch clamp device for recordinghERG currents. User-friendly software, excellent successrates, single additions or multiple additions of compoundto each cell and easy analysis result in reliable highquality data at an increased throughput with an
economical cost per data point.

Methods

1. Sanguinetti, M. C., et al., 1995. Cell. 81: 299-307
2. Brown, A.M. & Rampe, D. 2000. Pharm. News. 7(4): 15-Cells

20

CHO cells expressing hERG

3. Kirsch, G.E., et al., 2004. J. Pharmacol. Toxicol. Methods. 50: 93-101

Cell culture

4. Kang, J., et al., 2000. Eur. J. Pharmacol. 392: 137-40

Cells were cultured and harvested according to5. Zhou, Z., et al., 1999. J. Cardiovasc. Electrophysiol.

Nanion’s standard cell culture protocol.

10(6): 836-43
6. Wang, J., et al., 2003. Am. J. Physiol. Heart Circ. Physiol.

Electrophysiology

284: H256-H267

Whole cell patch clamp recordings were conducted

7. Chiu, P.J.S., et al., 2004. J. Pharmacol. Sci. 95: 311-319

according to Nanion’s standard procedure for the

8. Walker, B.D., et al., 1999. Br. J. Pharmacol. 128: 444-50

SyncroPatch® 384PE. A voltage step protocol from

9. Davie, C., et al., 2004. J. Cardiovasc. Electrophysiol.

-110 mV (holding potential) to +60 mV followed by a step

2004. 15(11):1302-9

to -40 mV was applied to the cells every 10 s for

10. Lacerda, A.E., et al., 2001. Eur. Heart J. Suppl. 3: K23-

pharmacology experiments. Peak amplitude at -40 mV

K30

was used for analysis.

11. Crumb, W.J. Jr., 2000. J. Pharmacol. Exp. Ther. 292(1):261-4

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960