Application Note

Channel:Cells:
Tools:

hNaV1.8CHO®
SyncroPatch 384PE

Stability and reproducibility of hNaV1.8
recordings on Nanion's SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich, Germany.
Cells were kindly provided by Charles River.

SummaryResults

The NaV1.8 gene (originally named PN3 or SNS; geneFigure

symbol SCN10A) encodes a voltage-gated sodiumincreasing voltage steps for an exemplar CHO cell(NaV) channel, selectively expressed in dorsal root

expressing hNaV1.8 and the corresponding current-ganglion (DRG) neurons . In contrast to the fast and1,21showscurrent

responses

tovoltage plot (normalized to the peak amplitude for each

rapidly inactivating TTX-sensitive channels, NaV1.8 is TTX-cell) for an average of 380 cells. hNaV1.8-mediatedvoltage-dependence of activation and inactivation2.

andhNaV1.8 is an interesting drug target for inflammatory

10andand neuropathic pain because modulation of this ionaBoltzmannchannel by inflammatory mediators appears to be a key

activation of -2.7 mV (n = 380), in good agreement withmechanism

the range found in the literature4-6.

ofDRG

nociceptor

sensitization

andactivation . Interestingly, the development of potent
3and selective NaV1.8 inhibitors has shown promising

currents

started

peak
20

to

activate

elicitedmV.

datawasTheequation

A-0.4

which records up to 384/768 experiments simultaneously
which helps to address this problem. It enables therecording of high quality data with reliable pharmacology,
and biophysical characterizations of the protein. Our
results show current-voltage relationships consistent
with published results4-6 and very stable recordings
using multi-hole chips. Furthermore, we show activation ofhNaV1.8 from different states results in altered compoundaffinity. We demonstrate the suitability of Nanion’s SyncroPatch 384PE for high throughput screening of hNaV1.8.

Download more Application Notes from www.nanion.de-0.6

0.5 nA
10 ms-40mV

betweenfittedausingVhalfofVoltage (mV)

-60 -40 -20-0.2

NaV1.8 inhibitors2.

Patch 384PE offers a high throughput gigaseal platform

revealing

Band this has fueled interest in the search for selectiveis often the electrophysiological assays. Nanion’s Syncro-about

wasresults in reducing neuropathic pain in animal models

The bottleneck for drug discovery involving ion channels

atresponse

2040 60

I/Imaxresistant and exhibits slower kinetics with a depolarized-0.8-1.0

Figure 1: A Raw traces from an exemplar cell expressing hNaV1.8 recordedon the SyncroPatch® 384PE. Shown are current responses to increasing
voltage steps from -60 to +60 mV. B Current-voltage plot for an average of380 cells. Shown are mean of peak amplitudes normalized to the maximumof each cell ± S.E.M.

Application NoteABCFigure 2: A Voltage protocol for the pharmacology experiments. C1 represents the peak current from the resting state and C2 represents peak current fromthe slow inactivated state. B Traces from an exemplar cell to the voltage protocol shown in A. Cursors for C1 (red) and C2 (green) are shown. C Screenshotof the data acquisition software for the SyncroPatch 384PE showing the time course of current amplitudes applying the two-step pulse protocol shown in A.
During this experiment, 302 of the wells received vehicle (0.25% DMSO) to monitor change in current over time. On the same plate, a 10-point concentration
response curve (each well received 1 concentration) for tetracaine was performed 6 times (shaded light blue rectangles), and two further controls: single
points for IC50 concentration (very light blue, 25 µM Tetracaine) and Full Block (dark blue, 250 µM Tetracaine) were used. The responses to tetracaine andDMSO only are highlighted on the right for 16 selected wells and for one at the bottom (DMSO control) in order to monitor the experiment. Compoundincubation time was 10 minutes. This highlights not only the stability of the recordings, but also the flexibility of experimental design using 384-well compoundplates.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application Note
NaV1.8 has traditionally been a difficult target to study

state, the voltage step protocol shown in Figure 2A wasdue to difficulty in expression in mammalian cell lines5.

devised. Figure 2B shows a recording from an exem-

Using the SyncroPatch 384PE and a CHO cell line

plar cell using this two-step voltage protocol. Using

stably expressing hNaV1.8, we were able to reliably

multi-hole (9X) chips, the peak current amplitude at C2

activation(slow inactivated phase) was consistently in the range

parameters consistent with hNaV1.8

recordhNaV1.8-mediatedcurrents

withat a high successof -1 to -3 nA, suitable for screening compounds on thisrate (99% for completed current-voltage plots; Figure 1).

ion channel. Figure 2C shows a screenshot of a typical

However, due to low current amplitudes using single hole

experiment highlighting the flexibility in experimental

chips (>80% of cells exhibited peak current at 10 mV ofdesign. On the plate, negative, positive, and single point<300 pA), multi-hole (9X) chips were used for the experi-

IC50 controls were performed along with concentration

4-6

ments which increased the success rate to >97% of cells

response curves.

exhibiting a peak amplitude of -1 to -3 nA.
Figure3shows3concentration

responsecurves

In order to assess the potency of compounds on thecalculated from different plates on different days. The

slow inactivated state of hNaV1.8 versus the resting

concentration response curves were calculated forA

BCD

Figure 3: Shown are 3 concentration response curves (A-C) calculated on different days using different plates and cell preparations. The peak current
amplitudes at C1 (resting state) and C2 (slow inactivated state; see Figure 2) were normalized to control and are shown. A Hill equation was fitted andthe calculated IC50 values at C1 (dark blue) and C2 (light blue) are shown for each plot. The IC50 is shifted at least 15-times to more potent values whenmeasured at C2 vs. C1 under these conditions. D The graph shows IC50 values obtained for tetracaine on different days and cell preparations for C1 (darkblue squares) and C2 (light blue circles). On average, IC50s were 34.8 ± 3.3 µM (n = 8 plates) for C1 and 1.5 ± 0.2 µM (n = 8 plates) for C2 as indicated by thedashed lines. Using these conditions, the shift was always larger than 15-fold and, importantly, the IC50 values vary very little across the plates.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax80636 Munich, Germany

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

Application Note
C1 and C2 for each well and the average concentra-preparations.

tion response curves were constructed for each plate.

amplitudes (normalized to control at the start of theImportantly, the IC50 values for tetracaine at C2, the slow

experiment) in negative (0.25% DMSO) or positive (250 µM

potent than at C1 under these conditions. The IC50 values

statistical parameter for evaluation and validation

calculated for C1 and C2 were consistent across plates.

ofThis is also shown in Figure 3D where the IC50 values arecalculated and revealed a Z’ value of 0.52 for thisplotted for 8 different plates. The values vary very little

assay with respect to the positive and negative controls,from the mean which is shown by the dashed line.

proving that this is an excellent assay for HTS7 of hNaV1.8.

inactivatedstate,

wereatleast

15times

moretetracaine)
highFigurecontrol

4Cshows

conditions.

throughput

screening

theThespreadZ(HTS)

offactor,data7,

awas

Figure 4D shows consistency of success rates at the end
consistentof the experiment (using quality control criteria I >200 pA

across wells, plates and on different days using

at the start of the experiment); full block using positive

different cell preparations. Figure 4A and B show that

control; RSeal >30 MΩ at the end of the experiment).

normalized peak current values for C1 and C2, respec-Success rates for completed experiments were typicallytively, are consistent across plates when experiments>90% and Z values >0.5.

are performed on different days using different cellAB289/302
Cells

279/302
Cells

amplitudes

278/302
Cells

were

290/302
Cells

288/302
Cells

291/302
Cells

0-100

300/302
Cells

279/302
Cells

291/302
Cells

291/302
Cells

291/302
Cells

290/302
Cells

0-1000C

500

1000
Number of Cells (n)1500

0Positive Control: Full Block 250µM Tetracaine (n = 17/18)

1000Z' = 0.52-100

Negative Control: DMSO 0.25% (n = 290/302)
050

100
150200
Number of Cells (n)250300

500

1000

1500

Number of Cells (n)D100
900.80.7

Success Rate

Z' Value% inhibition of current

100Success Rate at end of the exp. (%)% Inhibition of control currents(C1; 0.25% DMSO)

100current% Inhibition of control currents(C2; 0.25% DMSO)

Importantly,

80

0.6
Z'

70

0.5

60

0.4
1234Plate Number56Figure 4: Scatter plots showing % inhibition of current calculated at C1 (A) and C2 (B) after 10 min incubation in 0.25% DMSO using plates on different days
using different cell preparations. The stability of the recordings is consistent from day to day. B Calculation of the Z’ value for control recordings. Cells wereincubated in control solution (0.25% DMSO; light blue squares) or a full block concentration of tetracaine (250 µM; dark blue circles) for 10 minutes and% inhibition of current at C1 calculated with respect to the start of the experiment. The calculated Z’ value >0.5 reveals this to be an excellent assay forscreening hNaV1.87. D Success rate (light blue circles) at the end of the experiment based on quality control criteria (Ipeak > -200 pA at start; full block with
250 µM tetracaine; RSeal > 30 MΩ at end) for 6 different plates used on different days. The success rate was consistently > 90%. Also plotted are the Z’ values
for the same plates.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application Note
In conclusion, hNaV1.8 expressed in CHO cells canbe recorded on the SyncroPatch 384PE with a high®

successrate(typically>90%forhNaV1.8 and can be used to test compounds for statedependence. User-friendly software, excellent successcompletedrates, multiple additions of compound to each well,experiments using quality control criteria). The Z-factor, aavailability of single- or multi-hole chips and easy analysis

statistical parameter to evaluate and validate data

result in high quality, reliable data at a high through-for HTS7, reveals a value typically between 0.5 and 1,

put with an economical cost per data point. Additional

confirming that this is an excellent assay for HTS ofoptional extras such as temperature control and current

hNaV1.8 as the target ion channel. In order to increaseclamp increase the scope of experimental potential.

current amplitudes and success rates, multi-hole (9X)

chips were used so that current amplitudes in therange of -1 to -3 nA and success rates of >90% wereroutinely achieved. Importantly, there was little spreadof normalized current amplitudes and IC50 values

across the plate and between plates used on differentdays using different cell preparations. The SyncroPatch® 384PE

isa

highthroughput

andhighlyreliable automated patch clamp device for recording

References

Methods

1. Sangameswaran, L., et al., 1996. JBC. 271(11): 5953-Cells5956

CHO cells stably expressing hNaV1.8 (inducible cell line)

2. Theile, J.W. & Cummins, T.R., 2011. Front. Pharmacol.

were supplied by Charles River (Catalog #: CT6011).

2:54. doi:10.3389/fphar.2011.00054
3. Porreca, F., et al., 1999. PNAS. 96: 7640-7644

Cell Culture

4. Choi, J-S., et al., 2004. FEBS Letters. 572: 256-260

Cells were cultured and harvested according to

5. John, V.H., et al., 2004. Neuropharmacol. 46: 425-438

Nanion’s standard cell culture protocol. Cells were

6. Leffler, A., et al., 2007. JPET. 320: 354-364

induced to express NaV1.8 by incubation in tetracycline

7. Zhang, J-H., et al., 1999. JBS. 4: 67. doi: 10.1177/

for at least 24 hours prior to measurements.

108705719900400206
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch® 384PE using multi-hole chips. Currentvoltage recordings were made using voltage steps from-60 mV to 60 mV for 20 ms increasing in 10 mV steps, froma holding potential of -120 mV (leak subtraction protocolused).

Pharmacology

experiments

protocol to induce slow inactivation.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax

80636 Munich, Germany

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

usedtwo-step