Application Note

Channel:hNaV1.7
Cells:
CHO®
Tools:
SyncroPatch 384PE

Characterization of hNaV1.7 on Nanion's
SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich, Germany.
Cells were kindly provided by Anaxon.

Results

The NaV1.7 gene (SCN9A) encodes a voltage-gated100

peripheral nervous system. It has been isolated from rat

80

dorsal root ganglion (DRG) neurons , human medullary

60

nanomolar range1,2. The role of hNaV1.7 has yet to befully elucidated but is proposed to play an importantpart in nociception and pain sensing. NaV1.7 has been
implicatedtostates,particularin

hypersensitivity

playtoarolein

disease

pain

pain5

andinflammatory

heatfollowingburn(ndsable

NaV1.7 is a TTXs channel and is sensitive to TTX in the00>5

Seal Resistance (MΩ)

diTTX-sensitivity (TTX-resistant [TTXr], TTX-sensitive [TTXs]).

oNaV channels are in part characterized by their-5catc

the action potential is mediated by NaV channels.

00nervous systems. In particular, the fast upstroke of00

20of action potentials in the central and peripheral

10Different NaV channels play a key role in modulation

4000

thyroid cancer cells (hNE-Na)2 and PC12 cells3,4.

Start of Experiment
End of Experiment<11Cells (%)

sodium (NaV) channel, primarily expressed in theh)SummaryFigure 1:
Success rate (seal resistance) of CHO cells expressing hNaV1.7 on
the SyncroPatch® 384PE. Shown is a bar graph of seal resistanceson the SyncroPatch® 384PE at the start (light blue) and end (dark blue) ofthe experiment. Over 95% of the cells had a seal resistance >500 MΩ at thestart of the experiment and 91% at the end of the experiment.

injury6.

Common to many of the voltage-gated ion channels,
a number of compounds exhibit both state- anduse-dependence.

For

this

reason,itisimportant

to be able to reliably measure the effects of compounds
using different voltage protocols to investigate state anduse-dependency.
In
thethisApplication

SyncroPatch®

stablyexpressing

relationship

and

Note
384PE

present

data

using

characterizing

weCHO

cells

hNaV1.7.

thestate-Thecurrent-voltage

anduse-dependence

effects of the sodium channel blocker, tetracaine, areshown.
Download more Application Notes from www.nanion.de

Figure 2:
A Raw traces from an exemplar cell expressing hNaV1.7 recorded on theSyncroPatch® 384PE. Shown are current responses to increasing voltage
steps from -70 to +50 mV. B Current-voltage plot for an example cell.

Application NoteFigure 1 shows seal resistance values for CHO cells

using an inactivation voltage protocol. Tetracaine

expressing hNaV1.7 recorded on the SyncroPatch 384PE.

had a much larger effect on the 2nd (70% inactivated

The percentage of cells which had a seal resistance >500channels) peak shown in Panel A as compared withMΩ was 95% at the start of the experiment and 91% at thethe first peak. This indicates that tetracaine exhibits aend of the experiment. In this experiment, 356 cells werepreference for the inactivated state of NaV1.7 which®

usedinaforapharmacologicalsuccess

rateofexperiment

92%forresultingis confirmed by the concentration response curve.

completed

Tetracaine exhibits a much higher affinity for theexperiments. Figure 2 shows current responses toinactivated state of the receptor (IC50 = 370 nM, n = 356)

increasing voltage steps for an exemplar CHO cell
expressing NaV1.7 and the corresponding current-voltage
plot. NaV1.7 currents started to activate at about -50 mV
and peak response was elicited at around -20 mV.

compared with the open state (IC50 = 41.8 µM, n = 355,

Figure 5, 1st pulse). In this experiment a control wash step
is followed by addition of a single concentration of tetracaine and the concentration response curve is calculated across the whole plate. Additionally, after tetracaine
incubation, control solution is added in several wash
steps and the effect of tetracaine could be completelyreversed.

Figure 3:
A Current traces of an exemplar cell using an inactivation protocol toinactivate 70% of channels in the 2nd peak. B Online analysis showing peakcurrent of the 2nd peak versus time. Blue area indicates application oftetracaine. C Concentration response curve for tetracaine for an averageof 356 cells using a voltage protocol where 70% of NaV1.7 channels areinactivated. The IC50 for tetracaine on the inactivated state of the receptor
is 370 nM (n = 356).

Figure 3 shows a recording from an exemplar cell

Figure 4:
A Seal stability and B current stability for a representative 16 cells during arecording of CHO cells expressing hNaV1.7 recorded on the SyncroPatch®
384PE during an inactivation experiment. Shown is seal resistance(A) or current amplitude (B) versus time during a cumulative (3-point
concentration response curve (in this case 0.1% DMSO was added threetimes as a control). The seal resistance and current amplitude were stable
over the entire 18 minute recording.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application Note
Figure 4 shows seal stability and current stability over thetetracaine. In this experiment, a single concentration ofcourse of a 15 minute experiment. The current response

tetracaine

is shown during application of control (0.1% DMSO)

concentration response curve calculated across thesolution during an inactivation experiment. Both sealwhole plate. The current amplitude of the 1st, 2nd andresistance and current amplitude were stable over the10th pulses were used for analysis. The IC50 for tetracaine

entire 15 minute recording.

wasappliedtoeachcelland

thefor the 1st pulse was 41.8 µM (light blue), 2nd pulse (midblue) was 9.9 µM and for the 10th pulse (dark blue) was

3.0 µM (n = 355). The concentration response curve of
the 2nd and 10th pulses is shifted to the left comparedwith the first pulse showing that tetracaine exhibits usedependence. The success rate for completed experiments was 92%.
Figure 6 shows a screenshot of the SyncroPatch®
384PE

softwareexperiment.

A(PatchControl
color-coded384)

duringanoverview(basedonseal resistance in this case) of all 384 wells gives theuser a good impression of the success rate of theexperiment. The user can choose whether to visualize
raw traces or online analysis. In this case, online analysis
is chosen and the graphs represent current amplitudeof the 1st (light blue) and 10th (dark blue) voltage
pulse plotted against time. An individual well can behighlighted to monitor the progression of the experiment.
The online analysis shows the timepoint at which either
control solution (white) or tetracaine was applied (blue).

Figure 5:
A Current responses to a 10-pulse voltage protocol from 16 exemplar cells
expressing NaV1.7 recorded on the SyncroPatch® 384PE. The responses arein the presence of control solution (top left), 5 µM tetracaine (bottom left),
15 µM tetracaine (top right) or 50 µM tetracaine (bottom right). The peakcurrent responses of the 1st, 2nd and 10th pulses were used for analysisto investigate use-dependence. In this experiment, each cell received asingle concentration of tetracaine and the concentration response curve
was calculated across the whole plate. B Responses to a 10-pulse voltage
protocol of an exemplar cell in the presence of 15 µM tetracaine. C Current
amplitude of the 1st (light blue) and 10th (dark blue) peaks plotted against
time. D Concentration response curves to tetracaine for the 1st, 2nd and10th pulses. Average IC50’s are given.

Figure 5 shows current responses of 16 cells to a 10-pulse
voltage protocol and inhibition of NaV1.7 currents byNanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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

Application NoteFigure 6:
Graphical user interface of the screening and data analysis software used on the SyncroPatch® 384PE. Screenshot of depiction of online analysis data ofNaV1.7 expressing CHO cells as recorded on one NPC-384 patch clamp chip. Three hundred and eighty-four small color-coded pictures as seen in theupper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 500 MΩ), blue (Rmemb = 100–500 MΩ), light blueor 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 the 1st (light blue) and 10th (dark blue) pulse of a 10-pulse voltage protocol plotted against time of individual cells incontrol solution and then differing concentrations of tetracaine, 0, 5, 15 or 50 μM. Two wash steps with control solution prior to compound application wereperformed. For highlighted experiments, white indicates the wash solution and blue indicates the presence of tetracaine.

Exp end and chip
unload
8 - 12 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 - 2 control
washes + 3 compound concentrations

Analysis

Figure 7:
The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) for a 3-point concentration response curve plus 1 - 2 control washes
at the beginning of the experiment on NaV1.7 took approximately 15 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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

Application Note
Figure 7 offers a visual representation of a typicalexperiment on the SyncroPatch 384PE, from loading®

In conclusion, hNaV1.7 expressed in CHO cells canbe recorded on the SyncroPatch® 384PE with a highof the chip and priming of solutions, execution of thesuccess rate (> 90% for completed experiments). The

patch clamp experiment and analysis of the data.

SyncroPatch® 384PE is a high throughput and highly

Experiments can be performed either as a single point

reliable automated patch clamp device for recording

concentration

NaV1.7 and can be used to test compounds for state-response

curvewhereasingleconcentration of compound is applied to each cell andand use-dependence. User-friendly software, excellent

the concentration response curve calculated acrosssuccess rates, multiple additions of compound to eachthe whole plate, or cumulative concentration response

cell and easy analysis result in high quality, reliable data

curves can be performed on each cell. A complete

at a high throughput with an economical cost per dataexperiment which includes 1 - 2 control applications,point.

followed by 3 concentrations of compound takes
approximately 15 minutes. The SyncroPatch® 384PE
generates a throughput of 20,000 data points per daywith a competitive consumable cost per data point of~ €0.20.

References

Electrophysiology

1. Sangameswaran et al., 1997. JBC. 272 (3): 14805–14809

according to Nanion’s standard procedure for the

2. Klugbauer at al., 1995. Embo J. 14 (6): 1084-1090SyncroPatch® 384PE. Current-voltage recordings were

3. D’Arcangelo et al., 1993. J. Cell. Biol. 122 (4): 915-921

made using voltage steps from -70 mV to 50 mV for 20 ms

4. Toledo-Aral et al., 1997. PNAS. 94: 1527-1532

increasing in 5 mV steps, from a holding potential of

5. Nassar et al., 2004. PNAS. 101 (34): 12706-12711-120 mV (leak subtraction protocol used). Pharmacology

6. Shields et al., 2012. J. Neurosci. 32 (32): 10819-10832

experiments used voltage step protocols to mimic state-

Whole cell patch clamp recordings were conducted

and use-dependence.

Methods
Cells
CHO cells stably expressing hNaV1.7 were supplied byAnaxon.

Cell Culture
Cells were cultured and harvested according toNanion’s standard cell culture protocol.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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