Application Note

Channel: NaV1.5Peak /Late, hERG
Cells:
hiPSC-CM, CHO, HEK
Tools:
Port-a-Patch,
Port-a-Patch mini

Identification of cardiac liability in drug
discovery using the Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by NEXEL and Charles River, USA.
SummaryResults

In 2013 the Cardiac Safety Research Consortium

One goal of the CiPA initiative is to determine the(CSRC), the Health and Environmental Sciences Instituterequirements needed to deliver robust, reliable and(HESI), and the US Food and Drug Administration (FDA)

reproducible

ionchannel

proposed a paradigm to improve assessment of the pro-environment

using

higharrhythmic risk of therapeutic compounds. This paradigm,

automated patch clamp (APC) systems like the Syncro-

the Comprehensive In-vitro Proarrhythmia Assay (CiPA),Patch 384 or the Patchliner, low throughput systems like

was introduced to provide a more complete assessment

the Port-a-Patch family or manual patch clamp. Figureof proarrhythmic risk by evaluating and implementing

1 shows the different aspects of the CiPA initiative for acurrently

availablehighmulti-faceted approach to safety liability testing of com-importantpartthisofthroughput
isthemethods.

Anelectrophysiological

dataandinascreening

mediumthroughput

pounds.

evaluation of hERG, and also other cardiac channels
including NaV1.5 and CaV1.2. The Q&A draft from August

20201 describes how nonclinical assays such as patch
clamp can be used as a part of an integrated risk assessment prior to first-in-human studies, and in later stages ofclinical development.

Following up on hERG and NaV1.5 best practices andcalibration standards which have been publishedrecently2-4 on automated patch clamp devices, weshow here cardiac ion channel recordings from humaninduced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs) or overexpressing cell lines generated withthe world’s smallest patch clamp setups: Port-a-Patch
and Port-a-Patch mini. Recordings at RT or physiologicaltemperature of hERG recorded from HEK cells, and peakor late INa current recorded from iPSC-CMs or CHO cells

are shown. INa-Late was activated by ATX-II and blocked

by ranolazine, INa-Peak was blocked by tetracaine ina concentration-dependent manner, and hERG wasblocked by increasing concentrations of dofetilide.

Download more Application Notes from www.nanion.deFigure 1: The different aspects of the CiPA initiative for a more comprehensive assessment of cardiac safety testing of compounds.

Application Note

Human induced pluripotent stem cell-derived cardiomyo-dependent, binding with stronger affinity to the open andcytes (hiPSC-CMs) are becoming of increasing interest in

inactivated states compared with the resting state6. This

cardiac safety testing due to their relative abundance,

makes direct comparison with literature values complex.

their human origin and their recapitulation of nativeprotocol (Figure 2A), was enhanced by ATX-II and blocked

A-60

-40

by ranolazine (Figure 2B, C).

-20-2-95 mV-95 mV

C200 pA
10 ms

1 nA

Control
100 nM ATX II
100 µM Ranolazine

1 µM

100 nM ATX II
100 µM Ranolazine

INa (nA)-2

0.0-0.2-0.4-0.6-0.8
-1.0

1 nA
10 ms

10 µM

-3-4-5-6-7050

100150 200Time (s)Control
1 µM Tetracaine
10 µM Tetracaine
100 µM Tetracaine
300 µM Tetracaine

0100200Time (s)

300

1 nA

D

100 µM

020 ms60300 µM
-1-15 mV-100 mV-5C100 ms

INaV-Late(nA)B

40-4late (ramp)
40 mV 200 ms-15 mV 40 ms
notdisplayedpeak

20-1-3A

B

Voltage (mV)

Normalized blockLate), recorded during the ramp phase of the voltage

Ipeak (nA)human cardiomyocyte behavior. The late current (INa-5 ms

Tetracaine IC50 = 25.5 ± 5.7 µM (n = 6)
1.0 Hill = 1.00 ± 0.04
0.80.60.40.2
0.00.001 0.01

0.1110100[Tetracaine (µM)]

25050 ms

Figure 2: INa-Late recorded from hiPSC-CMs. A Voltage protocol usedfor NaV1.5 peak and late recordings. B INa-Peak and Late currents wererecorded using the protocol shown in A. INa-Late is shown enlarged in theinset. INa-Late was enhanced by ATX-II and blocked by ranolazine. C Timecourse of the experiment.

Peak NaV1.5 current (INa-Peak) was recorded from CHO

cells expressing NaV1.5 on the Port-a-Patch mini. Firstcurrents were recorded in response to increasing voltagesteps from -60 to +60 mV from a holding potential of -100
mV. A current-voltage plot was constructed and the data
fit with a Boltzmann equation revealing a Vhalf of activationof -32 mV (Figure 3A). Using a single voltage step protocol

to -15 mV repeated every 10 s, NaV1.5-mediated currents

were blocked by increasing concentrations of tetracaine(Figure 3B, C). The concentration response curve for an
average of 6 cells was constructed (Figure 3D) and an IC50of 25.5 ± 5.7 µM was calculated. This is in good agreementwith the range found in the literature5,6, although block ofNaV channels by local anaesthetics is strongly state-Download more Application Notes from www.nanion.deFigure 3: INa-Peak recorded from CHO cells. A Current-voltage plot of anexample CHO cell expressing NaV1.5. Responses to increasing voltage
steps are shown. B Block of NaV1.5 by increasing concentration of tetracaine. The top panel shows the single step voltage plot and the bottom
panel the traces from an example cell. C Corresponding timecourse of theexperiment. D Concentration response curve for tetracaine for an averageof 6 cells recorded on the Port-a-Patch mini.

Validation studies confirm that testing the effect of compounds on an increased number of human cardiac ionchannel currents including INa (NaV1.5 peak and late cur-rent) as well as IKr (hERG) leads to improved predictionof their clinical risk7,8. In addition to NaV1.5 expressed in acell line, we also investigated hERG expressed in HEK cells
using the Port-a-Patch or Port-a-Patch mini.

Application Note40 mV

B

500 ms
500 ms-40 mVDofetilide
1.0 IC50 = 161 ± 12 nM (n = 3)
Normalized Block-80mV

200 pA

Control
1 nM dofetilide
10 nM dofetilide

A40 mV-80 mVB

500 ms-80 mV

0.6

100 nM dofetilide
1 µM dofetilide
10 µM dofetilide

10 µM dofetilide

1.0

0.4

0.5

0.2

0.0

0.0
200 ms37°C

1.5

Control RT
Control 37°C
10 µM dofetilide (37°C)

0.8

Imax (nA)A

0.1

1Figure 4: hERG current recorded from HEK cells using the Port-a-Patchmini. A Raw data traces elicited using a standard 2-step voltage protocol(shown at the top) and block by increasing concentrations of dofetilide. B
Concentration response curve for an average of 3 cells. The data was fit
with a Hill equation revealing an IC50 = 161 ± 12 nM.

The Port-a-Patch mini was used to record hERG expressed
in HEK cells. The recordings were performed on the Port-aPatch mini at room temperature using a standard 2-stepvoltage protocol. The external solution was exchanged
manually using a pipette. In this way, low amounts ofcompound are required. Dofetilide blocked hERG
expressed in HEK cells at room temperature with an IC50of 161 ± 12 nM (n = 3), in good agreement with the range
found in the literature2,7.
In another set of experiments, the CiPA step-ramp voltage
protocol was used to elicit hERG expressed in HEK cells at
physiological temperature (37 ± 1°C) on the Port-a-Patch(voltage protocol is shown at the top of Figure 5A). In theseexperiments, the temperature controlled External Perfusion System for the Port-a-Patch was used to apply dofetilide. When the temperature of the bath solution was raisedto physiological temperature in control solution, the peakamplitude increased approximately 5 x compared to room
temperature (RT) (Figure 5). The currrent was then blocked
by 10 µM dofetilide.

00.2 nA

10100 1000 10000[Dofetilide (nM)]

200 ms100

200300Time (s)400500Figure 5: hERG current recorded from HEK cells using the Port-a-Patchand temperature controlled External Perfusion System A Raw data traces
elicited using a CiPA step-ramp voltage protocol (shown at the top) atRT, 37°C and following and block by 10 µM dofetilide. B Corresponding
timecourse of the experiment. Increasing the temperature of the bathsolution dramatically increased peak amplitude of hERG and the current
was completely blocked by 10 µM dofetilide.

In conclusion, INa-Peak and INa-Late could be reliably

recorded from hiPSC-CMs using the Port-a-Patch miniat room temperature. INa-Late which was activated by

ATX-II was blocked by external application of ranolazine.

Additionally, INa-Peak mediated by NaV1.5 expressedin CHO cells was reliably recorded and blocked bytetracaine in a concentration-dependent manner. hERG
expressed in HEK cells was recorded on the Port-a-Patchmini at RT and the current was blocked by increasing
concentrations of dofetilide. The Port-a-Patch with temperature-controlled External Perfusion System was used
to increase the temperature from RT to 37°C. This resulted
in a large increase in the current measured during theramp phase of the voltage protocol and the current wascompletely inhibited by a full block concentration ofdofetilide. Using the Port-a-Patch or Port-a-Patch mini,different voltage protocols can be used, in these experiments we used a single voltage step protocol for NaV1.5

recordings, along with a step-ramp protocol to enableboth peak and late current to be recorded in one sweep.

For hERG recordings we used a standard double voltage
step protocol and a step-ramp protocol recommended
by CiPA.

Download more Application Notes from www.nanion.deApplication Note
Nanion offers planar patch clamp systems with varying

strument with the highest throughput, SyncroPatch 384,

levels of throughput and automation. The Port-a-Patch

are valuable for applications in target identification, ef-and Port-a-Patch mini are miniaturized patch clampficacy, safety or HTS.

systems supporting giga-seal recordings from one
cell at a time. The devices offer fast and easy accessto high quality patch clamp data with only minimaltraining. Not only powerful research tools but also ideal for
educational purposes and evaluation of cells and ionchannels. The Patchliner and SyncroPatch 384 are fully
automated robotic systems for recording from up to 8 or
384 wells simultaneously. All devices are compatible withrecording ion channels important in safety screening as
suggested by the CiPA initiative9. This is possible in bothheterologous expression systems, e.g. HEK293 and CHO
cells, and potential cardiac model cells such as stem
cell-derived cardiomyocytes.
Nanion’s products are used in all aspects of drug screen-Figure 6: Accelerating Drug Discovery. Nanion’s products are used in all
aspects of drug screening, from basic research right through to lead optimization and safety testing.

ing and safety testing due to their ease-of-use coupledwith increased throughput or high throughput (HTS). The
whole portfolio from the smallest patch clamp rig in theworld, Port-a-Patch, to the automated patch clamp in-References

hERG (CT6001) were kindly supplied by Charles River,1. https://www.ema.europa.eu/en/documents/scientific-guide-

Cell culture

USA. Cardiosight-S® cells were kindly supplied by NEXEL.

line/ich-guideline-e14/s7b-clinical-nonclinical-evaluation-qt/
qtc-interval-prolongation-proarrhythmic-potential-questions-answers-step-2b_en.pdf. Accessed 02/03/2022
2. Brinkwirth, N., et al. 2020. JPTM 105: 106884

Cells were cultured and harvested according to Nanion’s standard cell culture protocols.
Electrophysiology

3. Han, X. et al. 2020. JPTM 105: 106890
4. Rotordam, MG., et al. 2021. JPTM 112: 107125

Whole cell patch clamp recordings were conducted

5. Finkel, A., et al. 2006. J. Biomol. Screen. 11: 488

according to Nanion’s standard procedure for the Port-

6. Li, H-L., et al. 1999. Mol. Pharmacol. 55: 134–141

a-Patch and Port-a-Patch mini. Currents were recorded

7. Kramer, J., et al. 2013. Sci. Rep. 3: 2100 https://doi.org/10.1038/

according to the CiPA protocol or according to standard

srep02100

protocols. NaV1.5 late currents were activated by

8. Crumb Jr, W.J., et al. 2016. JPTM. 81: 251-62.

application of 100 nM ATX-II in the external recording

9. Fermini, B., et al. 2016. J. Biomol. Screen. 21 (1): 1–11solution. Compounds were made as a 10 mM stock in
DMSO and diluted in external recording solution to theMethods

concentrations indicated.

Cells
CHO-NaV1.5 recombinant cell line (CT6007) and HEK-Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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