Application Note

Channels: hERG, NaV1.5, CaV1.2,Kir2.1, KvLQT1
Cells:
HEK, CHO, iPS-derived
cardiomyocytes
Tools:
SyncroPatch 384/768PE,
Patchliner, CardioExcyte 96

High Throughput Screening of Cardiac Ion
Channels
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

In 2013 the Cardiac Safety Research Consortium

CHO cells expressing hERG cells were recorded on the(CSRC), the Health and Environmental Sciences InstituteSyncroPatch 384PE with a high success rate (>80%).

(HESI), and the US Food and Drug Administration (FDA)

Figure 1 shows the current-voltage relationship for hERG.

proposed a new paradigm to improve assessment of theproarrythmic risk of therapeutic compounds. Until now,
drug safety testing has focussed on interaction with thehERG channel and QT prolongation which can lead topotentially fatal torsades de pointes (TdP). Although thisapproach has been largely successful in preventing newdrugs reaching the market with unexpected potential
to cause TdP, it is also possible that potentially valuable
therapeutics have failed due to this early screening. The
new paradigm, the Comprehensive In-vitro Proarrhythmia
Assay (CiPA) was introduced to provide a more
complete assessment of proarrythmic risk by evaluating
and implementing currently available high throughput
methods1,2. An important part of this remains electrophysiological evaluation of not only hERG, but also othercardiac channels including NaV1.5, CaV1.2, KvLQT1and Kir2.1. Additionally, new technologies, such as
impedance measurements, and cells such as stem cellderived cardiomyocytes, may provide useful tools forhigh throughput safety assessment.
Here we present high quality data with reliable
pharmacology on hERG expressing CHO cells, NaV1.5,
CaV1.2 or KvLQT1 expressed in HEK293 cells and Kir2.1

expressed in RBL cells on the SyncroPatch 384PE orPatchliner. Additionally, electrophysiological recordingson the Patchliner and Impedance measurements
ontheCardioExcyte

96ofstemcell-derived

cardiomyocytes are shown.
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Figure 1: Typical recording from hERG expressed in CHO cells on theSyncroPatch 384PE. The screenshot shows the data acquisition and analysissoftware used on the SyncroPatch 384PE. Wells are color-coded based onseal resistance (green: Rmemb > 500 MΩ, blue: Rmemb = 50 - 500 MΩ, lightblue or grey: Rmemb < 50 MΩ or disabled). Shown are current-voltage plots
for hERG-mediated currents elicited using a voltage step protocol from -120
mV to 40 mV increasing in 20 mV increments (after a depolarizing step to40 mV). B Raw traces from an exampel cell. The black trace highlights themaximum current in response to a test potential of -40 mV. C The currentvoltage relationship from an average of 322 cells.

Application Note
PharmacologyofhERG-mediatedcurrents

berecordedonthe384PE

highsuccessrate

SyncroPatchforcompletedcouldwithaexperiments.

Figure 2 shows the concentration response curves for four
known blockers of hERG with IC50 values as expectedin the literature. A summary of the IC50 values is given in
Table 1.

Voltage (mV)

-100 -60 -20

20 60-1.0
Figure 2: Average concentration response curves for 4 different hERG
blockers on the SyncroPatch 384PE. The concentration response curves
were constructed across the whole plate (single concentration per cell).
The SyncroPatch 384PE analysis software (DataControl 384) was used tocalculate the average concentration response curves, normalized tomaximum block and fitted with a standard Hill-equation. A summary of theIC50 values and success rates is shown in Table 1.

Figure 3 shows the current-voltage relationship of NaV1.5

expressed in HEK293 cells recorded on the SyncroPatch
384PE. An average activation and inactivation IV plot

I/Imax-0.2-0.6

D1.00.80.60.4
0.2
0.0-120 -100 -80 -60 -40
Voltage (mV)I/Imax

CFigure 3: NaV1.5 expressed in HEK293 cells recorded on the SyncroPatch
384PE. A The screenshot shows the data acquisition and analysis software
used on the SyncroPatch 384PE. Wells are color-coded based on seal
resistance (green: Rmemb > 500 MΩ, blue: Rmemb = 100 - 500 MΩ, light blue orgrey: Rmemb < 100 MΩ or disabled). Shown is the online analysis where peakcurrent is plotted against voltage to show the current-voltage relationship
B NaV1.5-mediated current responses to a voltage step protocol from-110 mV to 60 mV increasing in 10 mV increments. C Average current voltage
plot of 337 cells. The points were fit with a Boltzmann equation and the Vhalfof activation was -51 mV. D Average inactivation plot for 271 cells. Thepoints were fit with a Boltzmann equation and the Vhalf of inactivation was-84 mV.

is shown in Panels C and D, respectively. The Vhalf ofactivation was -51 mV and for inactivation was -84 mV
in good agreement with the literature3. Pharmacology of the NaV1.5 channel could be recorded on theSyncroPatch 384PE with success rates of up to 76% forcompleted experiments.
CompoundIC50 (nM)

Successrate (%)

Literature value(nM)Pimozide

5.9 (301)

78

1 - 184,5Astemizole

19.8 (315)

82

265

Cisapride33.1 (297)77

7- 264,5

Terfenadine

35.9 (321)84

7 - 1014,5

Table 1: IC50 values for pimozide, astemizole, cisapride and terfenadineon hERG-mediated currents recorded on the SyncroPatch 384PE. Shownare IC50 values (number of cells shown in brackets), success rate forcompleted experiments and the expected literature IC50 values. All IC50values recorded on the SyncroPatch 384PE agree well with the literature
values4,5.

Figure 4: Average concentration response curves for 4 different NaV1.5
blockers on the SyncroPatch 384PE. The concentration response curves
were constructed across the whole plate (single concentration per cell).
The SyncroPatch 384PE analysis software (DataControl 384) was used tocalculate the average concentration response curves, normalized tomaximum block and fitted with a standard Hill-equation. A summary of theIC50 values and success rates is shown in Table 2.

Nanion Technologies GmbHphone + 49 89 219 095 0

Ganghoferstr. 70Afax80339 Munich, Germany

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

Application NoteFigure 4 shows the concentration response curves for

The 2nd phase (plateau) of the cardiac action potential isfour sodium channel blockers on NaV1.5-mediatedmediated by the NaV1.5 and CaV1.2 channels10. CaV1.2-110 mV. At more depolarized holding potential the IC50Patchliner (Figure 6). The current was blocked by theas expected6. The IC50 values and success rates aremanner with an IC50 of 12.8 ± 8.0 nM (n = 3) in good agree-currents. In these experiments holding potential wasfor lidocaine increased by a factor of 35 (data not shown)summarised in Table 2.

has been recorded in the voltage clamp mode on theCaV1.2 blocker, nifedipine, in a concentration-dependent
ment with the literature11. Action potentials recorded fromstem cell-derived cardiomyocytes on the Patchliner wereCompoundIC50 (µM)

Successrate (%)

Literature value(µM)

Amitriptyline

5.0 (251)

65

1.67

Mexiletine

97.3 (241)63498

Lidocaine

295 (291)

763536

Tetracaine

4.5 (264)

69

9.39

Table 2: IC50 values for amitriptyline, mexiletine, lidocaine and tetracaine
on NaV1.5-mediated currents recorded on the SyncroPatch 384PE. Shown
are IC50 values (number of cells shown in brackets), success rate for completed experiments and the expected literature IC50 values.

The K+ current IK1, encoded by the Kir2.1 gene, is responsible for the final phase of the cardiac action potentialalso shortened by nifedipine (Figure 6C). Current clamp
measurements at high throughput will soon be possible on
theSyncroPatch

384PE.

Stemcell-derived

cardiomyocytes offer a distinct advantage over adultventricular myocytes given the relative ease with whichlarge quantities can be produced and their provensuccess

onautomated

patch

clamp

systems12,13.

However, these cells must be properly characterized andcompared with adult ventricular cardiomyocytes before
their use in safety screening will become routine.
ABVoltage [mV]
0-80 -60 -40 -20

20Kir2.1 channel is expressed endogenously in RBL cells and-1

has also been successfully recorded on the SyncroPatch
384PE. Figure 5 shows Kir2.1 channels activated by-2
1 nA

addition of external solution containing high K+.

-3

25 ms
C

1.0 Nifedipine (n = 3)

Normalised block

0.8

IC50 = 12.8 ± 8.0 nM

0.6
0.4

Imax [nA]and for maintaining the resting membrane potential10. The

DControl
10 µM Nifedipine

0 mV

0.2
0.0

1 pM 100 pM 10 nM 1 µM
[Nifedipine]-75 mV

20 mV
100 msFigure 6: CaV1.2 expressed in HEK cells recorded on the Patchliner. A Current
responses to a voltage step protocol (from -80 mV to +30 mV for 100 ms
Figure 5: Kir2.1 endogenously expressed in RBL cells recorded on the increasing in 10 mV increments) of an exemplar cell expressing CaV1.2.
SyncroPatch 384PE. Kir2.1 was activated by addition of external solution B corresponding IV curve. Vhalf of activation was -8.8 mV. C Concentration
containing high K+ concentration. Solution exchange rate is in the order of response curve for nifedipine for an average of 3 cells. IC50 was 12.8 ± 8.0 nM
50 ms on the SyncroPatch 384PE.
(n = 3). D Action potential recorded from a stem cell-derived cardiomyocyte
recorded on the Patchliner. The action potential amplitude was reduced
and the duration was shortened by nifedipine.

Nanion Technologies GmbHphone + 49 89 219 095 0

Ganghoferstr. 70Afax80339 Munich, Germany

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

Application NoteA500
400
300
200200 pA

100100 ms-100-50
500Voltage (mV)

Figure 7: KvLQT1 expressed in HEK cells recorded on the Patchliner.
A Current responses to a voltage step protocol of an exemplar cell
expressing KvLQT1. B Corresponding IV plot.

Repolarization of the cardiac action potential, or phase3, is facilitated by IKs which is encoded by the KvLQT1/Impedance [Ohm]B
Imax (pA)AB
2.0

Nifedipine
0.50nM
0.03nM-0.5
1.010nM
-1.0
0.5
30nM
100nM -1.5
0.0300nM
0.0
0.4
0.8
0.0 0.2 0.4 0.6
Time [s]Time [s]1.5

Figure 8: Dose-dependent effect of nifedipine on the impedance andthe EFP. Compounds were applied as indicated to a monolayer of
20 k Cor.4U cells. The mean beats, derived from 10 s sweeps, are shown
in A for the impedance and in B for the EFP. Included is the influence on
the impedance amplitude and pulse-width (arrows in A), as well as on theextracellular field potential duration (FPD; arrow in B).

mink genes10. The KvLQT1 channel has been successfullyrecorded on the Patchliner (Figure 7).

In

conclusion,

NanionTechnologies

offershighthroughput automated patch clamp systems which areRecent advances in technologies such as impedancecompatible with recording all ion channels important inoffer the ability to measure effects of compounds onsafety screening as suggested by the CiPA initiative. This

the contractility of cardiac cells. Since impedanceis possible in both heterologous expression systems, e.g.

measurements can be made on cultured cells and stem

HEK293 and CHO cells, and potential cardiac model cells

cell-derived cardiomyocytes at high throughput and atsuch as stem cell-derived cardiomyocytes. Additionally,physiological temperature, this technique may provethe CardioExcyte 96 is an impedance system capable

a useful complementary technology to patch clampof testing compounds on the contractility and EFP ofelectrophysiology. Figure 8 shows the effect of the CaV1.2

contractile cells such as Cor.4U cells shown here.

blocker, nifedipine, on the impedance and extracellular
field potential (EFP) duration using the CardioExcyte 96.

References

Methods

1. https://www.ilsiextra.org/hesi/science/cardiac/cipa/SitePages/Home.

Cells

aspx

CHO cells expressing hERG, HEK293 cells expressing

2. Sager, P.T., et al., 2014. Am. Heart J. 167(3): 292-300

NaV1.5, CaV1.2 or KvLQT1, RBL or stem cell-derived
cardiomyocytes

3. Sheets, M.F. & Hanck, D.A. 1999. J. Physiol. 514.2: 425-436
4. Kirsch, G.E., et al., 2004. J. Pharmacol. Toxicol. Methods. 50: 93-101
5. Kang, J., et al., 2000. Eur. J. Pharmacol. 392: 137-40

Cell culture

6. Bean, B.P., et al., 1983. J.Gen. Physiol. 81: 613 - 642

Cells were cultured and harvested according to7. Harmer, A.R., et al., 2011. Br. J. Pharmacol. 164: 260–273

Nanion’s standard cell culture protocols.

8. Wang, D.W., et al., 1997. J. Clin. Invest. 99 (7): 1714–1720
9. Finkel, A., et al., 2006. J. Biomol. Screen. 11(5): 488-496Electrophysiology

10. Hoekstra, M., et al., 2012. Front. Physiol. doi: 10.3389/fphys.2012.00346

Whole cell patch clamp recordings were conducted

11.Liao, P., et al., 2007. J. Biol. Chem. 282: 35133-35142

according to Nanion’s standard procedures for the12. Stoelzle, S., et al., 2011. Front. Pharmacol.

doi: 10.3389/SyncroPatch 384PE and Patchliner.

fphar.2011.00076
13. Stoelzle, S., et al., 2011. J. Biomol. Screen. 16: 910–6

Nanion Technologies GmbHphone + 49 89 219 095 0

Ganghoferstr. 70A

fax80339 Munich, Germany

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