Application NoteChannel:
Cells:
Tools:

hNaV1.5
CHO; iCell®
Cardiomyocytes2Patchliner

INa-Late recorded from CHO cells andhiPSC-CMs on Nanion´s Patchliner
The electrophysiology team at Nanion Technologies GmbH, Munich.
iCell® Cardiomyocytes2 kindly provided by Fujifilm Cellular Dynamics International.
CHO cells kindly provided by Charles River.
SummaryResults

The voltage-gated Na+ channel 1.5 (NaV1.5) is encoded

CHO cells expressing NaV1.5 were used on the Patch-of the cardiac action potential (AP). The NaV1.5 channel

was used to elicit INa-Late as recommended by the CiPA

subunits1. When the cardiac cell membrane depolarizes,

the protocol was used for analysis. ATX-II was used toNaV1.5 opens for a short time allowing an influx of Na

activate INa-Late. Figure 1 shows the raw current traces ofDuring the action potential, these channels can recoverlidocaine. Figure 2 shows the concentration responseby the SCN5A gene and is responsible for the rising phaseis comprised of a pore-forming a subunit and auxillary b+ions resulting in the upstroke of the action potential.

liner. The voltage protocol shown at the top of Figure 1study. The peak inward current during the ramp part ofINa-Peak and INa-Late in the presence of ATX-II and block byfrom inactivation and re-open resulting in a sustained

curve to lidocaine for the peak (A) and late (B) current.

current termed INa-Late. Although this current is substantially

The IC50 was approximately 7 times lower on the latesmaller than the peak Na current (INa-Peak), it is active+during the plateau phase and therefore contributes toAP morphology2. There is a growing body of evidence
that increased INa-Late can have a pathophysiological rolecurrent compared with the peak current.
40 mV, 200 ms-15 mV, 40 ms

100 ms-80 mV-120 mV

0.5 nAin acquired heart diseases such as myocardial ischemia

50 ms

and heart failure . INa-Late is elevated in several pathological
3conditions which could result in Na+-overload in thesecells. A number of loss or gain-of-function mutations inControl (60 nM ATX-II)
30 µM Lidocaine
2 mM Lidocaine (full block)the SCN5A gene have been identified which lead tochanges in the magnitude or duration of INa-Peak or INa-Lateresulting in fatal arrhythmias1. INa-Late is a potential drug

target to treat cardiac disorders such as angina, heart
failure and arrhythmia4. It is also an important target insafety pharmacology as enhancement of INa-Late is proarrhythmic5.

0.5 nA

50 pA
10 ms

10 ms

In this study the Patchliner was used to record INa-Late fromCHO cells and hiPSC-CMs. INa-Late was recorded using thevoltage protocol specified by CiPA and activated using
ATX-II. INa-Late could be recorded from CHO cells stably
expressing NaV1.5 and blocked by lidocaine. INa-Late could
also be detected in iCell® Cardiomyoctes2 and blockedby ranolazine.
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Figure 1:
INa-Late was activated by 60 nM ATX-II using the voltage protocol shown at
the top. The current was blocked by lidocaine. A single concentration oflidocaine was applied per well followed by full block by 2 mM lidocaine(red). Recordings performed at 35°C. The insert shows the peak (left) andlate (right) current enlarged.

Application Note
NaV1.5-peak (Vhold = -110 mV)
Max = 1, IC50 = 171.5 µM, Hill = 0.98

AB

NaV1.5-late: (n = 17)
1.0 Max = 0.7, IC50 = 23.6 µM, Hill = 1.4
0.8

Normalized block

1.0

Normalized blockA
0.8

Control

0.6

0.6

100 nM ATX II
100 µM Ranolazine

0.4

0.4
0.2

0.2

0.0

100 µM

B-100-200-200-400-300-400

significant for n = 5 cardiomyocytes.
In conclusion, the Patchliner, in combination with
anhNaV1.5expressing

CHOcelllineezinlano10010myocyte2 showing activation by ATX-II and block byranolazine. The current elicited by ATX-II was statistically*II8000INa-Late current recorded from an example iCell® Cardio-400600Time (s)TX200Ra0*

A-600

µM

selective blocker, ranolazine. Figure 3 shows INa-Peak andC0l

Cardiomyocytes2. The INa-Late current was blocked by the100 µM RanolazinenM

detect a small but statistically significant INa-Late in iCell®

100 nM ATX II0INa-Late (pA)

Figure 1, and ATX-II at a concentration of 100 nM, we could

20 ms

50 ms

We also investigated whether we could record INa-Late in

hiPSC-CMs. Using the same voltage protocol as shown in

0.1nA

2 nA

tro

Figure 2:
A Concentration response curve for lidocaine on NaV1.5 peak current andB late current. Single point concentration response curves were performed
and the concentration response curves calculated over several plates.
The IC50 for lidocaine was approximately 7 times lower on the late current
compared with the peak current.

on1 µM
10 µM[Lidocaine]C

100 nMINa-Late (pA)

0.0
100 nM 1 µM 10 µM 100 µM 1 mM 10 mM[Lidocaine]

Figure 3:
A Example traces from an iCell® Cardiomyocyte2 showing INa-Peak and INa. B Timecourse of the experiment. C Average current amplitude for 5
Latecells. INa-Late was statistically significantly enhanced by ATX-II and blocked byranolazine (P<0.05, student’s paired t test).

(CharlesRiver) or hiPSC-CMs, is a robust electrophysiological toolfor investigating effects of compounds on INa-Late for drug
discovery and safety testing.

References

Cell culture

1. Han D., et al., 2018. Exp. Biol. Med. 243: 852-863.
2. Horvath B., & Bers D.M. 2014. ESC Heart Failure. 1: 26–40.
3. Maier L.S., & Sossalla S. 2013. JMCC. 61: 44-50.
4. Makielski J.C. 2016. Trends Cardiovasc Med. 26(2): 115–

CHO cells and hiPSC-CMs were cultured and harvested
according to Nanion‘s standard cell culture protocols forcell lines and stem cells, respectively.
Electrophysiology

122.

Whole cell patch clamp recordings were conducted

5. Belardinelli L., et al. 2013. JPET. 344: 23–32.

according to Nanion’s standard procedure for thePatchliner with standard solutions. Currents were elicited

Methods

using a voltage step/ramp protocol (see Figure 1)
repeated every 10 s in the presence of 60 nM (CHO) orCells

100 nM (hiPSC-CMs) ATX-II applied via the external

CHO cells stably expressing hNaV1.5 (Catalog # CT6007)

solution. Experiments were performed at 35°C (CHO) or

were supplied by Charles River.

room temperature (hiPSC-CMs). The peak currentiCell® Cardiomyocytes2 (Catalog # CMC-100-012-000.5)

recorded during the ramp phase of the voltage protocol

were supplied by Fujifilm Cellular Dynamics International.

was used for analysis of INa-Late.

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