Application Note

Channel:Cells:
Tools:

hNaV1.9
HEK293
SyncroPatch 384

High Throughput Pharmacology of NaV1.9
Channels on Nanion’s SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Icagen, Inc., USA.

SummaryResults

The SCN11A gene encodes the voltage-gated sodium

Currents

channel NaV1.9 which is predominantly expressed inrecorded on the SyncroPatch 384. Figure 1 shows the bio-ganglia (DRG) and trigeminal ganglia1-3. NaV1.9 isAn average activation and inactivation IV plot is shownslowdorsal

activation

rootwith

littledepolarization near the resting membrane potential
generating a persistent, tetrodotoxin (TTX)-insensitive
current

which

inactivates

properties

suggestmediatedby

amplificationofNaV1.9

that

only
the

mainlydepolarizing

slowly .
2

Theseconductancecontributes

responses

toto
sub-

threshold stimuli leading to lower action potential (AP)firing thresholds and increase in AP firing frequency .
3-5

atory and neuropathic pain6. Several gain-of function
mutations in SCN11A have been identified which result

both in good agreement with literature values2,3,7,10,11.
AB
0-50-1000

10 20 30 40 50time (ms)C-10050-50-1.0

development of novel pain therapeutics.

-1.5

a high throughput collected on the SyncroPatch 384.
Biophysical properties of NaV1.9 expressed in HEK cells(cells kindly provided by Icagen, Inc., USA) and concentration response curves for three NaV channel blockers
are shown, including use-dependence of tetracaine.

Download more Application Notes from www.nanion.de10 msDV (mV)Given its proposed role in pain perception, NaV1.9 has

pharmacology on hNaV1.9 expressing HEK293 cells atreliably-43 mV (n = 341) and for inactivation was -41 mV (n = 240),-0.5

Here we present high quality data with reliablebein panel C and D, respectively. The Vhalf of activation wasin either painful neuropathy7 or an insensitivity to pain8,9.
gained some attention as a potential target for thecould

physical properties of hNaV1.9 expressed in HEK293 cells.

The role of hNaV1.9 has yet to be fully elucidated butis proposed to be involved in nociception of inflamm-hNaV1.90.5 nA

byofby1.0I/Imax

characterizedneurons

I (nA)

sensory

Amplitude (mV)small-diameter

mediated

0.5

0.0-100-50V (mV)

0Figure 1: Biophysical properties of NaV1.9 recorded on theSyncroPatch 384. Currents were obtained by application of a voltage
step protocol from a Vhold of -120 mV. Voltage steps started at-100 mV to 40 mV increasing in 10 mV increments. B NaV1.9-mediated current
responses to the voltage step protocol shown in A. C Average current
voltage plot of 341 cells. The points were fit with a Boltzmann equation andthe Vhalf of activation was -43 mV. D Average inactivation plot for 240 cells.
The points were fit with a Boltzmann equation and the Vhalf of inactivation
was -41 mV.

Application Note
Pharmacology of the hNaV1.9 channel could besecond pulse (mid-blue) was 0.8 μM and for the tenthrates of up to 83% for completed experiments. Fig-racaine for the first pulse was 11.7 μM (dark blue), sec-

performed on the SyncroPatch 384 with successpulse (light blue) was 0.7 μM (n = 104). The IC50 for tet-ure 2 shows the concentration response curves forond pulse (mid-blue) was 8.0 μM and for the tenth pulse

three(light blue) was 2.7 μM (n = 100). While the concentra-NaV

channelblockers

on

NaV1.9-mediatedcurrents. In these experiments holding potential wastion response curves for T-CN-1752 overlap almost exactly-120 mV. The IC50 values are summarised in Table 1.

(Fig. 3C), the curves for tetracaine of the second and the-0.5

pulse (Fig. 3D), showing that tetracaine exhibits use-de-1.0 IC = 335.0 ± 0.3 µM
50(n = 342)

pendence whereas T-CN-1752 does not. The success rate
for completed experiments was around 80%.
A

200 pA
20 ms-1.5-30 mV
50 mV

Ipeak

0.5-1.00.0

400
600time (s)10-6

1.0 IC = 1.1 ± 0.1 µM
50(n = 148)

D
1.0 IC = 15.6 ± 0.5 µM
50(n = 145)

0.50.5

Relative Inhibition of INa

C

200 ms10-5 10-4 10-3
Lidocaine (M)200 msC10-910 10 10 10
T-CN-1752 (M)-8-7-6-5-0.5

200

10 10 10 10 10 10Tetracaine (M)-9-8-7-6-5-4

400 600time (s)D
C1: IC50 = 0.8 ± 0.2 µM
C2: IC50 = 0.8 ± 0.1 µM
C3: IC50 = 0.7 ± 0.1 µM

0.00.0control Tetracaine Full Block-1.0

Relative Inhibition of INa

200

B

Ipeak (nA)

FullBlock

0.2 nAVehicletenth pulse are shifted to the left compared to the firstB
Relative Inhibition of INa

A1.0

n = 104 (81%)

C1: IC50 = 11.7 ± 1.7 µM
C2: IC50 = 8.0 ± 1.5 µM
C3: IC50 = 2.7 ± 0.4 µM

1.0

n = 100 (78%)

Figure 2: Inhibition of NaV1.9 by lidocaine, T-CN-1752 and tetracaine recorded
0.50.5on a four-hole NPC-384 patch clamp chip A NaV1.9 mediated currents wereactivated by depolarizing cells from -120 mV to -30 mV. Average current
response after application of vehicle is indicated in blue, grey trace shows
the response after 300 µM tetracaine (full block). Average current amplitudes± SEM of 135 wells were plotted over time. B-D The concentration response
0.00.0curves for lidocaine (B), T-CN-1752 (C) and tetracaine (D) were constructed-8-7-6-5-410-7 10-6 10-5 10-41010101010
across multiple wells (single concentration per well). The SyncroPatch 384
T-CN-1752 (M)Tetracaine (M)analysis software (DataControl 384) was used to calculate the average
concentration response curves, normalized to maximum block and fitted
Figure 3: Effect of high frequent stimulation on potency of tetracaine blockwith a standard Hill-equation.
of NaV1.9. A Current response to a 10-pulse voltage protocol from one
exemplar cell expressing hNaV1.9 recorded on the SyncroPatch 384. TheIn the experiment in Figure 3, a 10-pulse voltage protoresponses are shown in the presence of control solution (black trace) andcol was used to investigate use-dependence of inhibition 5 μM tetracaine (blue trace). The peak current responses of the 1st, 2nd and10th pulses were used for analysis to investigate use-dependence. In thisof NaV1.9 currents by lidocaine and tetracaine. A single
experiment, each well received a single concentration of tetracaine orconcentration of each compound was applied to each T-CN-1752 and the concentration response curve was calculated acrossmultiple wells. B Current amplitude of the 1st (black), 2nd (dark blue) and 10th
well and the concentration response curve calculated(light blue) peaks plotted against time. C, D Concentration response curves
across multiple wells. The current amplitude of the first, for T-CN-1752 (C) and tetracaine (D) for the 1st, 2nd and 10th pulses.

second and tenth pulses were used for analysis. The IC50

for T-CN-1752 for the first pulse was 0.8 μM (dark blue),

Download more Application Notes from www.nanion.deApplication NoteFigure 4: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of online analysis dataof hNaV1.9 expressing HEK293 cells as recorded on a multi-hole (4x) NPC-384 patch clamp chip. Three hundred and eighty-four small color-coded pictures
as seen in the upper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 120 MΩ), blue (Rmemb = 25 – 120MΩ), light blue or grey (Rmemb < 25 MΩ or cells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayedon the right. Graphs show current amplitudes of NaV1.9 channels during the voltage step to -30mV during application of control solution (white region),compound (blue region) and full block by 300 µM tetracaine (dark grey region).

Exp end and chipunload
5 min

10 - 12 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 - 2 control washes,
1 compound addition, 1 full block addition

Analysis

Figure 5: Timeline of an experiment on the SyncroPatch 384. The completion of one experiment on the SyncroPatch 384 patch clamp chip (384 wells) for asingle point or cumulative concentration response curve on NaV1.9-mediated currents took approximately 17-20 min.

Download more Application Notes from www.nanion.deApplication Note
Compound

IC50 (µM)

Success
rate (%)

Literature
value (µM)

T-CN-1752

1.1 (148)

76

1.610

Tetracaine

15.6 (145)693210

Lidocaine

335 (342)

8935610caine and grey is full block with 300 µM tetracaine.
In conclusion, hNaV1.9 expressed in HEK293 cells

Figure 4 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 successrate of the experiment. The user can choose whether tovisualize raw traces or online analysis. Here, online analysis
is chosen and the graphs represent current amplitude
time.

Anindividual

wellcanbehighlighted

to monitor progression of the experiment. In theOnline

Analysisview,

thetimepoints

this example, white shows incubation in control solution,
blue is different concentrations of T-CN-1752 or tetra-

Table 1: IC50 values for T-CN-1752, tetracaine and lidocaine on NaV1.9mediated currents recorded on the SyncroPatch 384. Shown are IC50values (number of wells shown in brackets), success rate for completed
experiments and the expected literature IC50 values.

vs.

vertical lines, as well as different background colors. Inatwhich

can be recorded on the SyncroPatch 384 with highsuccess rates for completed experiments (>70%). The
IC50s of three NaV channel blockers were in good agreement the literature10. Furthermore, we observed use-dependent block of tetracaine on NaV1.9.
The SyncroPatch 384 is a high throughput and highly
reliable automated patch clamp device for recording
NaV1.9 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.

solution additions have been made are indicated by

References

Methods

1. Dib-Hajj, S.D., et al. , 1998. Proc. Natl. Acad. Sci. U.S.A.

Cells

95: 8963–8968.

HEK293 cells expressing hNaV1.9 kindly provided by

2. Dib-Hajj, S.D. ,et al., 1999. FEBS Lett. 462: 117–120.

Icagen, Inc.

3. Dib-Hajj, S.D., et al. , 2002. Trends Neurosci. 25: 253–259.
4. Dib-Hajj, S.D., et al., 2015. Nat. Rev. Neurosci. 16(9): 511-

Cell culture

9.

Cells were cultured and harvested according to

5. Herzog, R. I., et al., 2001. J. Neurophysiol. 86: 1351–1364.

Nanion’s standard cell culture protocol.

6. Baker, M.D. & Wood, J.N., 2001. Trends Pharmacol. Sci.
22: 27–31.

Electrophysiology

7. Huang, J., et al., 2014. Brain. 137: 1627–1642.

Whole cell patch clamp recordings were conducted

8. Leipold, E., et al., 2013. Nat. Genet. 45: 1399–1404.

according to Nanion’s standard procedure for the

9. Huang, J., et al., 2017. J. Clin. Invest. 127: 2805–2814.

SyncroPatch 384 and multihole (4x) chips. A voltage step

10. Lin, Z., et al., 2016. PLoS One. 11(8): e0161450.

protocol from a holding potential of -120 mV to -30 mV

11. Zhou, X., et al., 2017. Front Pharmacol. 22: 8:852.

was applied to the cells every 20 s for pharmacology
experiments. Peak amplitude at -30 mV was examined
for analysis (unless otherwise indicated).

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