Application Note

Channel:Cells:
Tools:

NaV1.5
CHO & HEK
SyncroPatch 384i

NaV1.5-ΔKPQ late INa current properties andpharmacology on the SyncroPatch 384i
Theelectrophysiology

team

atNanion

Technologies

GmbH,Munich.

Cells kindly provided by Metrion Biosciences.

SummaryResults

The cardiac late Na current (late INa) generates persistent

HEK cells expressing NaV1.5-ΔKPQ and CHO cells express-action potential. Several mutations in the SCN5A genewith good success rates. Peak currents with an amplitude

currents throughout the plateau phase of the cardiacing NaV1.5-WT were recorded on the SyncroPatch 384i

cause a form of hereditary long QT syndrome (LQT3)1-3.

of -1.6 ± 0.1 nA (n = 219) were recorded from WT cells andThe ΔKPQ mutation deletes residues Lys 1505, Pro 1506

-5.0 ± 0.1 nA (n = 310) from NaV1.5-ΔKPQ cells using single

and Gln 1507, resulting in a sustained, non-inactivating
current during long (over 50 ms) depolarizations . This1,2

hole plates. The late INa current was negligible in WT cells

using the step-ramp voltage protocol shown in Figure 1

sustained current causes prolongation of the action(-35 ± 1.8 pA, n = 58) but was reliably recorded using thepotential which can result in fatal ventricular arrhythmias

same protocol from the NaV1.5-ΔKPQ cells (-176 ± 63 pA,such as Torsade de Pointes (TdP) .
1

One aim of the Comprehensive In Vitro Pro-arrhythmia
Assay (CiPA) initiative is to improve drug safety testingn = 74; QC parameters: Rseal >400 MΩ, late INa >-100 pA)
using single hole chips. The late INa current was increased
and, therefore, success rate improved using multi-holein pre-clinical development by evaluating the pro-NPC-384 chips with 4 holes per well (4x). Using this typearrhythmic risk of a compound4,5. Validation studies

of chip, late INa current amplitude was increased to -405

channels using the toxin ATX-II or veratridine, or using a-120 mV

enhancers and test compounds.
Here we present data collected on the SyncroPatch 384i showing the peak and late INa current recorded from WT and NaV1.5-ΔKPQ cell lines. Peak current could be reliably recorded from both cell types. InWT cells, late INa was negligible in the absence of ATX-II,

whereas the late INa from NaV1.5-ΔKPQ cells could be reliably recorded. Peak current from WT, and peak and late

INa from NaV1.5-ΔKPQ was inhibited by ranolazine andmexiletine and IC50 values agreed well with the literature6.
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CHO NaV1.5 WT
HEK NaV1.5∆KPQ (1x)HEK NaV1.5∆KPQ (4x)

QKP
5∆
1.
V-100
INa Late (pA)

reduce the risk of cross-reactions between late-current

1.

0cell line with LQT3 mutations in NaV1.5 without the needfor pharmacological enhancement. The latter might also

5∆KPQ-80 mVV-15 mVHE
(4 K Nx) a

their clinical risk. Late INa can be recorded in WT NaV1.540 mV

1.
5

as well as IKr (hERG) leads to improved prediction oflate INa >-200 pA).

Vcurrents including INa (NaV1.5 peak and late current)± 14 pA, n = 167; Figure 1; QC parameters: Rseal >250 MΩ,HE(1 K Nx) aincreased number of human cardiac ion channelCW HOTNaconfirm that testing the effect of compounds on an-200-300

1 nA50 ms-400-500
Figure 1: Current recorded from WT and NaV1.5-ΔKPQ expressing cells. A
Raw current traces from CHO NaV1.5-WT (black), and NaV1.5-ΔKPQ (blue)
using either single hole (1x) or multi-hole (4x) chips. B Bar graph showing
current amplitude of late INa from WT and NaV1.5-ΔKPQ. Late INa wasnegligible in WT cells but could be reliably recorded in NaV1.5-ΔKPQ.

Application Note

As the ΔKPQ mutation affects the voltage-dependenceof NaV1.5 channels it was important to determine thebiophysics of this cell line on the SyncroPatch 384i platform. Activation and inactivation of peak currents ofNaV1.5-ΔKPQ were recorded using voltage-step protocols. The activation plot is shown in Figure 2 and fit witha Boltzmann equation. The V0.5 of activation of the mean

Compounds known to block NaV1.5 were applied to assess

their effect on NaV1.5-ΔKPQ peak and late currents. Concentration response curves for mexiletine and ranolazine

are shown in Figures 4 & 5, respectively. IC50 values aresummarized in Table 1. Both mexiletine and ranolazine

blocked the late current with a lower IC50, i.e. higher potency, than the peak current as expected6.

current IV was -34.5 mV (n = 252) in good agreement withMexiletine
NaV1.5∆KPQ late IC50 = 4.5 ± 0.3 µM (n = 72)
NaV1.5∆KPQ peak IC50 = 48.2 ± 4.8 µM (n = 110)

the literature2,7. The inactivation plot is shown in Figure 3and fit with a Boltzmann equation. The V0.5 of inactivation was -75.2 mV (n = 296), also in good agreement with

1.0and ionic composition of solutions.
-60-40-20204060

20 ms
10-7-5

1 nA
2 ms

V0.5Act = -34.5 mV (n = 252)40-1.0

IPeak (nA)

-0.5

10-20 µM
3 µM
12 µM
35 µM
118 µM360 µM

0.80.6100 pA
50 ms

0.4
0.2-1.5

1 nA
20 ms

0.0-2.0

10-7-2.5
V0.5Inact = -75.2 mV (n = 296)

10-3

1.0Normalized block

20-20

10-510-4[Mexiletine (M)]

RanolazineNaV1.5∆KPQ late IC50 = 11.9 ± 1.1 µM (n = 74)
NaV1.5∆KPQ peak IC50 = 80.0 ± 5.9 µM (n = 94)

Voltage (mV)-40

10-6Figure 4: Inhibition of peak and late currents mediated by NaV1.5-ΔKPQ bymexiletine. Mexiletine was applied in a cumulative manner from lowest
concentration to highest concentration on each well. Concentration
response curves for peak current (black circles) and late current (bluetriangles) were constructed and are shown for an average of 72 wells (peak)and 110 wells (late) including the average current traces. Multi-hole chips (4holes per well) were used.

Figure 2: Current-voltage plot of activation of NaV1.5-ΔKPQ. The average
current-voltage plot for n = 252 wells is shown for NaV1.5-ΔKPQ. The plot is fit
with a Boltzmann equation which reveals a Vhalf of activation of -34 mV. Thetraces for an exemplar well are also shown in the inset.

-60

1 nA

0.0-4-80

50 ms

0.2
IPeak (nA)-3100 pA

0.4-1-20.8

0.6

Voltage (mV)
-80

Normalized block

the literature1,7 when taking into account differences inexperimental protocol including Vhold, pre-pulse duration

0 µM
1 µM
3.5 µM
12 µM
35 µM
118 µM

0.2 nA

5 ms

Figure 3: Current-voltage plot of inactivation of NaV1.5-ΔKPQ. The average
current-voltage plot for inactivation for n = 296 wells is shown for NaV1.5ΔKPQ. The plot is fit with a Boltzmann equation which reveals a V0.5 ofinactivation of -75.2 mV. The traces for an exemplar cell are also shownin the inset.

Download more Application Notes from www.nanion.de10-61010[Ranolazine (M)]
-5-4

10-3

10-2

Figure 5: Inhibition of peak and late currents mediated by NaV1.5-ΔKPQ byranolazine. Ranolazine was applied in a cumulative manner from lowest
concentration to highest concentration on each well. Concentration
response curves for peak current (black circles) and late current (bluetriangles) were constructed and are shown for an average of 74 wells (peak)and 94 wells (late) including the average current traces. Multi-hole chips (4holes per well) were used.

Application Note

Figure 6: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384i. Screenshot depicts online analysis (peakamplitude) of NaV1.5-ΔKPQ-expressing HEK cells as recorded on one NPC-384 patch clamp chip. At the start of the experiment, 1 vehicle application ismade followed by 5 concentrations of ranolazine or mexiletine with an incubation time of 3.5 mins per concentration. At the end of the experiment, afull-block concentration of tetracaine (333 µM) was added to each well. As a control, part of the chip received only vehicle. Multi-hole chips were used
where 4 holes were present per well. The data of the 384 well plate representation in the upper left part are color-coded for easy assessment of data.
Depending on the seal resistance, pictures are green (Rmemb > 100 MΩ), blue (Rmemb 50 - 100 MΩ), light blue (Rmemb <50 MΩ) or grey (well disabled).
One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show online analysis data (peakamplitude) of NaV1.5-ΔKPQ in increasing concentrations of either ranolazine or mexiletine. Using the voltage-step protocol, IC50s can be calculated for bothpeak and late current in each well using a single voltage protocol.

Exp end and chipunload
5 min

25 - 30 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control application (vehicle), 5
compound additions (3.5 mins per concentation), full-block
concentration (tetracaine) at the end of the experiment

Analysis

Figure 7: Timeline of an experiment on the SyncroPatch 384i. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a
cumulative concentration response curve with control application, cumulative additions of compound and full-block on NaV1.5-ΔKPQ-mediated currents
took approximately 32 - 37 mins.

Download more Application Notes from www.nanion.deApplication Note
NaV1.5-WT
IC50 (µM)

NaV1.5-ΔKPQ
IC50 (µM)

by 5 concentrations of ranolazine or mexiletine inLiterature
IC50 (µM)

increasing

CompoundPeak

Late

Peak

Late

Peak

Late*Ranolazine

143 ± 9(63)-80.0 ± 5.9(94)11.9 ± 1.1(74)79.5 ± 5.5(12)16.7 ± 1.7(5)Mexiletine

83 ± 5(83)-48.2 ± 4.8(110)4.5 ± 0.3(110)21.9 ± 2.6(10)12.2 ± 1.1(12)Table 1: IC50 values for ranolazine and mexiletine block of peak currentof NaV1.5-WT (single hole chips) or peak and late current of NaV1.5-ΔKPQ(4 hole chips) and comparison with literature values6. * in the presenceof ATX-II. Values are given as mean ± S.E.M, number of wells in brackets.

Figure 6 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 thesuccess rate of the experiment. The user can easily
toggle between raw traces and online analysis. In theexample

shown,

onlineanalysisvaluesarechosen where peak amplitude in increasing concentrations of ranolazine or mexiletine are shown. In theseexperiments, 1 control application of vehicle followed

concentrations

wasapplied(3.5mins

incubation per concentration) and finally a full-block
application of tetracaine was added. Concentration
response curves were calculated for each well. The cells
were stable for at least 30 minutes. An individual well canbe highlighted to monitor progression of the experimentand is shown enlarged at the bottom of the screen.
In conclusion, peak and late INa could be reliably

recorded from NaV1.5-ΔKPQ expressing HEK cells without
pharmacological enhancement which could potentiallyinteract with test compounds. Late INa from NaV1.5-ΔKPQ
was more potently inhibited by ranolazine and mexiletine
than peak NaV1.5-ΔKPQ or WT peak currents. Using theSyncroPatch 384i automated patch clamp system incombination with the NaV1.5-ΔKPQ cell line provided areliable high throughput CiPA-compliant cardiac safety

screening assay without the need for openers such as
ATX-II.
Electrophysiology

References

Whole cell patch clamp recordings were conducted

1. Bennett, et al., 1995. Nature. 376: 683-685
2. Wang et al., 1996. PNAS. 93: 13200–13205
3. Wang, et al., 1995. Cell, 80: 805-811
4. Fermini, et al., 2016. JBS. 21(1): 1-11, doi:10.1177/108705
7115594589
5. Cavero & Holzgrefe. 2014. Expert Opin. Drug Saf. 13(6):745-758
6. Guo & Jenkinson. 2019. JPTM. 99: 106575, doi: 10.1016/j.
vascn.2019.04.001
7. Abdelsayed, et al., 2017. J. Physiol. 595.18: 6165–6186

according to Nanion’s standard procedure for theSyncroPatch 384i using single hole or multi-hole (4 holesper well) chips. A voltage step to -15 mV was used torecord peak current, and a voltage ramp from 40 mV to-80 mV over 100 ms was used to record late INa current.

Holding potential was -80 mV. For pharmacology
experiments, compounds were applied in increasing
concentrations to each well (incubation time: 3.5 mins
per concentration) followed by a full-block concentrationof tetracaine (333 µM). Concentration response curves
for individual wells were constructed with minimum fixed

Methods

to 0 and maximum fixed to 1. Average concentration

Cells

wells for each compound using DataControl 384. Mean

response curves were then constructed for all successful

HEK cells expressing NaV1.5-ΔKPQ were kindly providedby Metrion Biosciences. CHO cells expressing NaV1.5-WTand S.E.M was calculated from the individual IC50 values.
Data was only included in the analysis when it satisfied

were also used.

QC parameters: Rseal >400 MΩ, late INa >-100 pA for single

Cell culture

hole chips.

hole chips and Rseal >250 MΩ, late INa >-200 pA for multi-Cells were cultured and harvested according toNanion’s standard cell culture protocol.

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