Application NoteChannel:
Cells:
Tools:

hNaV1.7
CHO
Port-a-Patch®

Patch clamp recordings of hNaV1.7 on
Nanion’s Port-a-Patch®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Anaxon.

SummaryResults

The NaV1.7 gene (SCN9A) encodes a voltage-gatedFigure 1 shows the raw current responses to aperipheral nervous system and has been isolated from rat

expressing hNaV1.7 recorded on the Port-a-Patch®. The

sodium (NaV) channel, primarily expressed in thevoltage step protocol from an exemplar CHO celldorsal root ganglion (DRG) neurons , human medullary

average activation curve for an average of 16 cells

1

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

is also shown. NaV1.7 currents started to activate atabout -40 mV, peak response was elicited at around
Different NaV channels play a key role in modulation

0 mV and Vhalf of activation was -11 mV (n = 16).

of action potentials in the central and peripheral
nervous systems. In particular, the fast upstroke ofNaV channels are in part characterized by theirAB
Test Potential (mV)TTX-sensitivity (TTX-resistant [TTXr], TTX-sensitive [TTXs]).
NaV1.7 is a TTXs channel and is sensitive to TTX in the-60 -40 -20-0.2

fully elucidated but is proposed to play an important-0.4

nanomolar range1,2. The role of hNaV1.7 has yet to berole in nociception and pain sensing. NaV1.7 has been

implicated to play a role in disease pain states, inparticular inflammatory pain5 and hypersensitivity to heatfollowing burn injury . Common to many of the voltage6-0.6

0.5 nA
5 ms20 40I/Imax

the action potential is mediated by NaV channels.

-0.8

gated ion channels, a number of compounds display ahigher affinity for the inactivated state of the channel. For
this reason, it is important to be able to reliably record
both activation and inactivation kinetics of the channel.
Inthisusing

theApplication

Note

Port-a-Patch®

wepresent

characterizing

CHO

datacells

stably expressing hNaV1.7. The hNaV1.7 activation andinactivation properties and TTX sensitivity are consistent
with those reported in the literature1,2,7,8.

Download more Application Notes from www.nanion.de

Figure 1:
A Raw current traces of an exemplar CHO cell expressing hNaV1.7 recordedon the Port-a-Patch®. Currents were elicited using a voltage step protocol
from a holding potential of -120 mV to -60 mV for 20 ms increasing in 10
mV steps up to 50 mV. B Corresponding IV activation plot for an averageof 16 cells.

Application NoteFigure 2 shows current responses of an example cell toCurrent responses in the absence and presence of TTX

an inactivation voltage protocol. A 5 s pre-pulse to differ-

are shown in Figure 3. A concentration response curve

ent test potentials was followed by a step to 0 mV for 10

was constructed revealing an IC50 = 43 ± 7 nM (n = 5), intion plot for an average of 5 cells is also shown. The Vhalfof inactivation was -73 mV (n = 5), in good agreement

good agreement with the literature2.

A

B

with the literature1,2,7,8.

AControl
3nM
10nM
30nM
100nM
300nM
1µM
WashoutB
0.8
I/Imax0.6
0.4

0.5 nA

0.2

0.5 nA
2 ms

0.0-120 -80 -40 040
Test Potential (mV)

Figure 2:
A Raw current traces of an exemplar CHO cell expressing hNaV1.7 to an
inactivation protocol. Currents were elicited using a voltage step protocol
to -120 mV for 5 s increasing in 10 mV steps, followed by a step to 0 mV
for 10 ms. Currents at 0 mV are shown. B Relative peak amplitude at 0 mV
was plotted against the test potential. Shown is an inactivation plot for an
average of 5 cells. Points were fitted with a Boltzmann equation. Vhalf ofinactivation was -73 mV.

NaV1.7 is a TTX-sensitive channel and TTX was appliedusing the external perfusion system for the Port-a-Patch®.

Relative Blockms. Shown are current responses at 0 mV. The inactiva-

1.0
0.8
0.6
0.4
0.2
0.0TTX
IC50 = 43 ± 7 nM (n = 5)

0.1

15 ms

10 100 1000
[TTX(nM)]

Figure 3:
A Raw current traces of hNaV1.7 and subsequent block by TTX recorded on
the Port-a-Patch. Shown are raw current traces from an exemplar CHO cell
expressing hNaV1.7, elicited using a voltage step protocol from a holding
potential of -120 mV to 0 mV for 20 ms, and increasing block by increasing
concentrations of TTX. B Concentration response curve for block of hNaV1.7
by TTX for an average of 5 cells. IC50 = 43 ± 7 nM (n = 5).

In

conclusion,hNaV1.7

expressedinprovidedbyAnaxon

canbe

on

Port-a-Patch®

withactivation,

theCHOreliably

cells

recordedinactivation

properties and TTX sensitivity as expected1,2,7,8.
Cell culture

References

Cells were cultured and harvested according to
1. 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 the Porta-Patch®. 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 to 0 mV for 20 ms, then back to -120 mV,indicated (-120 mV to 30 mV in 10 mV increments)

Anaxon.

Pharmacology experiments used a voltage step protocol
sweep interval 3 s (P/4 leak subtraction used).

Nanion Technologies GmbHphone +
 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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