Application Note

Channel:Cells:
Tools:

hNaV1.5
HEK293
SyncroPatch 384PE

High Throughput Pharmacology of NaV1.5
Channels on Nanion’s SyncroPatch 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by EMD Millipore, USA.

SummaryResults

The NaV1.5 channel, encoded by the SCN5A gene,

For the evaluation of the performance of hNaV1.5

skeletal muscle and heart1. It is TTX insensitive with an IC50

Resistanceis a voltage-gated sodium (NaV) channel found inin the micromolar range1. NaV1.5 is responsible for the(HEK293) cells, Seal Resistance, Cslow and the Series(Rseries)

weredetermined

fromoneexperiment (Fig. 1) .

upstroke of the cardiac action potential in both

repolarization4.

B 40

to exhibit state- and use-dependence when acting on

30

approximately 30 times lower at depolarized holding
potentials where inactivation was almost complete5. For
this reason, it is important to test potency of compounds
at different holding potentials.
Here we present high quality data with reliable
pharmacology on hNaV1.5 expressing HEK293 cells at ahigh throughput collected on the SyncroPatch 384PE.
Current-voltage plots and concentration response curves
for four NaV channel blockers are shown, including
lidocaine at different holding potentials.

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

Local anaesthetics, such as lidocaine, have been shown
the cardiac sodium channel5. The IC50 was shown to be00

100

compound safety screening.

>5edsabldicardiac function, it has become an important target inMΩ0Given the importance of this channel in maintainingΩcardiac

Mmodify

Seal Resistance
C 60
4020201000Cslow(pF)51
10 0-115 5-220 0-25>25can00

inactivation

20<5cardiac arrythmias3,4. Furthermore, effects of NaV1.540-5conduction velocity, thus causing potentially dangerous

60

Cells (%)

effects on the rate of cardiac deploarization andMΩ

electrocardiogram (ECG) and can have profound8000

can lead to prolongation of the QRS interval of thestart of experimentend of experiment<1action potential in human heart. Block of this channel

A100

<551
10 0-115 5-220 0-25>25

critical for generation and propagation of the cardiac

Cells (%)

ventricular and atrial myocytes2 and is therefore

Rseries (MΩ)

Figure 1: Statistics of hNaV1.5 expressing HEK cells recorded on one NPC384 chip on the SyncroPatch® 384PE
A Success rate (seal resistance) of individual HEK cells on the SyncroPatch
384PE. Shown is a bar graph of seal resistances at the start (light blue) andend of the experiment (dark blue). B Bar graph of Cslow and C Rseries
values for HEK cells expressing hNaV1.5.

Application Note
Currents

mediatedbyhNaV1.5

could

bereliably

recorded on the SyncroPatch 384PE with a high successrate. Figure 2 shows the current-voltage relationship ofhNaV1.5 expressed in HEK293 cells recorded on the SyncroPatch 384PE. An average activation and inactivation IV

plot is shown in Panels C and D, respectively. The Vhalf ofactivation was -51 mV and for inactivation was -84 mV ingood agreement with the literature6.

Figure 3: 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 1.

-120 mV; cumulative measurement (IC50: 275 µM)-120 mV (IC50: 279 µM)

I/Imax-100-60 -20 20 60-1.0

I/Imax

-0.2
-0.6

1.0
0.8
0.6
0.4
0.20.0-120-100 -80 -60 -40
Voltage (mV)

Figure 2: 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.

Pharmacology of the hNaV1.5 channel could be-100 mV (IC50: 74 µM)-80 mV (IC50: 8 µM)
1.0
Relative Inhibition of Peak INa

C Voltage (mV) D

0.80.60.40.20.0100performed on the SyncroPatch 384PE with successrates of up to 76% for completed experiments. Figure 3 shows the concentration response curves forfourNaVchannelblockersonNaV1.5-mediatedcurrents. In these experiments holding potential was-120 mV. The IC50 values are summarised in Table 1.

101

102

103

104

Lidocaine [µM]
Figure 4: Effect of holding potential on potency of lidocaine block of NaV1.5.
The concentration response curves were constructed at different holding
potentials (as indicated) either using a single concentration of compoundper cell (solid lines) or cumulative concentration response curves (dashedline). The IC50 for lidocaine was shifted by a factor of 35 when holding
potential was changed from -120 mV to -80 mV.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70

fax

80339 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 NaV1.5 expressing HEK293 cells as recorded on one NPC-384 patch clamp chip. Three hundred and eighty-four small color-coded pictures as seenin 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
the right. Graphs show current amplitudes of NaV1.5 channels during the voltage step to -10mV during application of control solution (white region) andinhibition by lidocaine (blue region).

Exp end and chipunload
5 - 13 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 - 2 control
washes + 1 - 5 compound concentrations

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) for asingle point or cumulative concentration response curve on NaV1.5-mediated currents took approximately 12-20 min.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70fax

80339 Munich, Germany

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

Application Note
CompoundIC50 (µM)

Success
rate (%)

Literature
value (µM)

Amitriptyline 5.0 (251)

65

1.67

Mexiletine

97.3 (241)63498

Lidocaine

295 (291)

763535

Tetracaine

4.5 (264)

69

9.39

vs.

wasinfluencedbyholdingtime.

An

individual

wellcanbehighlighted

to monitor progression of the experiment. In theOnline

Analysisview,

thetimepointsatwhichsolution additions have been made are indicated byTo investigate whether the potency of lidocaineNaV1.5

visualize raw traces or online analysis. Here, online analysis
is chosen and the graphs represent current amplitude

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.

onrate of the experiment. The user can choose whether topotential,

concentration response curves were constructed atdifferent holding potentials (Fig. 4). As expected5, theIC50 for lidocaine was shifted by a factor of >30 using
a holding potential of -80 mV vs. -120 mV. The IC50 was
not changed when the experiments were performed as

cumulative (dashed line) or single point concentration
response curves (solid line) at -120 mV.

vertical lines, as well as different background colors. In
this example, white shows incubation in control solution
and blue is lidocaine.
In conclusion, hNaV1.5 expressed in HEK293 cells canbe recorded on the SyncroPatch 384PE with highsuccess rates for completed experiments (>70%). The
IC50’s of four NaV channel blockers were in good agreement the literature5-9 and the IC50 for lidocaine was influenced by holding potential5.

The SyncroPatch 384PE is a high throughput and highly
reliable automated patch clamp device for recording

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 success

NaV1.5 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.

References

Methods

1. Gellens, M.E., et al., 1992. PNAS. 89: 554-558

Cells

2. Nerbonne, J.M. & Kass, R.S. 2005. Physiol. Rev. 85: 1205-HEK293 cells expressing hNaV1.5.

1253
3. Bowes, J., et al., 2012. Nat. Rev. Drug. Disc. 11: 909-922

Cell culture

4. Erdemli, G., et al., 2012. Front. Pharmacol. doi: 10.3389/Cells were cultured and harvested according tofphar.2012.00006

Nanion’s standard cell culture protocol.

5. Bean, B.P., et al., 1983. J.Gen. Physiol. 81: 613 - 642
6. Sheets, M.F. & Hanck, D.A. 1999. J. Physiol. 514.2: 425-

Electrophysiology

436

Whole cell patch clamp recordings were conducted

7. Harmer, A.R., et al., 2011. Br. J. Pharmacol. 164: 260–

according to Nanion’s standard procedure for the273

SyncroPatch 384PE. A voltage step protocol from a

8. Wang, D.W., et al., 1997. J. Clin. Invest. 99 (7): 1714–holding1720indicated) to -10 mV was applied to the cells every 5 s for

9. Finkel, A., et al., 2006. J. Biomol. Screen. 11(5): 488-496

pharmacology experiments. Peak amplitude at -10 mV

potential

was used for analysis.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70

fax

80339 Munich, Germany

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

of-120mV(unlessotherwise