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Application NotehNaV1.7
CHOPatchliner

Pharmacology on hNaV1.7 performed onNanion’s Patchliner at Vhalf
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Anaxon.

SummaryResults

The NaV1.7 gene (SCN9A) encodes a voltage-gatedFigureperipheral nervous system and has been isolated from rat

hNaV1.7.

1showstheactivation

andinactivation

sodium (NaV) channel, primarily expressed in thecurves for an average of 8 CHO cells expressing

dorsal root ganglion (DRG) neurons1, human medullaryabout -40 mV, peak response was elicited at aroundthyroid cancer cells (hNE-Na)2 and PC12 cells3,4.

NaV1.7currents

startedtoactivate

at-10 mV and Vhalf of activation was -19 mV (n = 8).
The Vhalf of inactivation was -74 mV (n = 8), in good

Different NaV channels play a key role in modulationagreement with the literature1,2,7,8. TTX was applied reveal-nervous systems. In particular, the fast upstroke ofthe literature2.

NaV channels are in part characterized by their

TTX-sensitivity (TTX-resistant [TTXr], TTX-sensitive [TTXs]).
NaV1.7 is a TTXs channel and is sensitive to TTX in theA-60 -40

nanomolar range1,2. The role of hNaV1.7 has yet to be-0.6

role in nociception and pain sensing. NaV1.7 has been

particular inflammatory pain and hypersensitivity to heat
5C

this reason, it is important to be able to reliably measure
the effects of compounds at Vhalf of inactivation, thevoltage at which 50% of the channels are inactivated.
In this Application Note we present data using an8-channelPatchliner®

characterizing

CHOcellsstably expressing hNaV1.7. The hNaV1.7 activation andinactivation properties are consistent with those reported
in the literature1,2,7,8. The potency of sodium channel
blockers

mexiletine,

tetracaine,

amitriptyline

andlidocaine were compared using a holding potential of-120 mV vs the Vhalf or inactivation.
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Relative Blockhigher affinity for the inactivated state of the channel. For

2040-0.8

following burn injury6. Common to many of the voltagegated ion channels, a number of compounds display a-20-0.2-0.4

fully elucidated but is proposed to play an important

implicated to play a role in disease pain states, inB

Test Potential (mV)TTX
1.0 IC50 = 20 ± 9 nM
(n = 6)
0.8 Vhold = -120 mV
0.6

1.0
0.8

I/Imaxthe action potential is mediated by NaV channels.

ing an IC50 = 20 ± 9 nM (n = 6), in good agreement withI/Imaxof action potentials in the central and peripheral

0.6

0.4
0.2
-120 -100 -80 -60 -40
Test Potential (mV)

0.4
0.2
0.0

10-11 10-10 10-9 10-8 10-7 10-6[TTX (M)]

Figure 1:
A Average current-voltage plot of activation, Vhalf of activation was-19 mV (n = 8). B Average inactivation plot, Vhalf of inactivation
was -74 mV (n = 8). C Concentration response curve for TTX(Vhold = -120 mV) reveals an IC50 = 20 ± 9 nM (n = 6) in good agreementwith the literature2.

Application Note
protocol from -120 mV to 0 mV and inhibition of thecurrent

byincreasing

concentrations

oftetracaine. The timeplot of the experiment is also shown.
BControl
1 µM
10 µM
100 µM1 mM

500 pA

5 ms

Peak Current (nA)A

0.00 1µ10µ 100µ1m-1.0

The concentration response curves for 4 known NaVblockers

at0.6

different

holding

potentials

are shown in Figure 3. The Vhalf was determined

BVhalf (n = 7)
IC50 = 1.2 ± 0.1 µM

0.6
0.4
0.2
10-910-7
10-510-3[Tetracaine (M)]0.0

10-9 10-7 10-5 10-3
[Lidocaine (M)]

DVhalf (n = 7)
IC50 = 467 ± 193 nM-120 mV (n = 2)
IC50 = 8.4 µM

1.00.8Vhalf (n = 4)
IC50 = 14 ± 5 µM
-120 mV (n = 3)
IC50 = 138 ± 47 µM

0.60.60.40.40.20.20.0-120 mV (n = 2)
IC50 = 354 µM

0.80.20.8Vhalf (n = 7)
IC50 = 9.2 ± 4.9 µM

1.0-120 mV (n = 6)
IC50 = 11 ± 2 µM

0.4

1.0

100 200 300 400 500 600
Time (s)Figure 2:
A Raw traces from an exemplar cell recorded on the Patchliner® showing
inhibition of hNaV1.7 current by increasing concentrations of Tetracaine.
Shown are current responses to a voltage step protocol from -120 mV to0 mV for 20 ms. B Timeplot of the experiment.

channel0.8C-2.0-3.0

1.00.0Relative BlockhNaV1.7A
Relative Block

Figure 2 shows current responses to a voltage step

0.0
101010[Amitriptyline (M)]10-9-7-5

10-9-3

10-7
10-3
10-5
[Mexiletine (M)]

Figure 3:
Average concentration response curves for A Tetracaine, B Lidocaine
C Amitriptyline, and D Mexiletine at Vhold = Vhalf (black) andVhold = -120 mV (blue), IC50 values are shown on individual plots.

and set individually for each cell. The IC50s for eachcompound are shown on each individual graph using
two different holding potentials (Vhalf or -120 mV). In allcases, holding the cells at Vhalf caused a leftward shift ofthe concentration response curve.

Inconclusion,

providedbyhNaV1.7

Anaxonexpressed
canbeinreliably

CHO

cells

recordedon the Patchliner® with activation and inactivation
properties as expected1,2,7,8 and compound potencies
can be reliably measured at Vhalf.

Cell culture

References

Cells were cultured and harvested according to1. Sangameswaran et al., 1997. JBC. 272 (3): 14805–14809

Nanion’s standard cell culture protocol.

2. Klugbauer at al., 1995. Embo J. 14 (6): 1084-1090
3. D’Arcangelo et al., 1993. J. Cell. Biol. 122 (4): 915-921

Electrophysiology

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

Whole cell patch clamp recordings were conducted

5. Nassar et al., 2004. PNAS. 101 (34): 12706-12711
6. Shields et al., 2012. J. Neurosci. 32 (32): 10819-10832
7. Vijayaragavan et al., 2001. J. Neurosci. 21 (20): 7909-7918
8. Cummins et al., 2004. J. Neurosci. 24 (38): 8232-8236

according to Nanion’s standard procedure for thePatchliner®. Current-voltage recordings were made using
voltage steps from -60 mV to 50 mV for 20 ms increasingin 10 mV steps, from a holding potential of -120 mV.

Methods

Inactivation protocol used a 5 s pre-pulse to the voltage

Cells

followed by a step to 0 mV for 10 ms, 20 s sweep interval.

CHO cells stably expressing hNaV1.7 were supplied byfrom -120 mV or Vhalf (set individually for each cell) toindicated (-120 mV to 30 mV in 10 mV increments)

Anaxon.

Pharmacology experiments used a voltage step protocol
0 mV for 20 ms, then to -120 mV for 2 s then back to-120 mV or Vhalf, interval 10 s (P/4 leak subtraction used).

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