Application Note

Channel:Cells:
Tools:

rNaV1.8
ND7-23Patchliner®

Characterization of rNaV1.8 (ND7-23) onNanion´s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Millipore.

SummaryResults

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

Figure 1 shows current responses to increasing voltage

channel, selectively expressed in dorsal root ganglion

the corresponding activation and inactivation curves forsymbol SCN10A) encodes a voltage-gated sodium (NaV)

steps for an exemplar ND7-23 cell expressing NaV1.8 and(DRG) neurons. DRGs transmit peripheral stimuli to thean average of 19 cells. NaV1.8 currents started to acti-central nervous system and are involved in nociception.

vate at about -40 mV, peak response was elicited ataround 20 mV and Vhalf of activation was -9 mV (n = 19).

of DRG action potentials. In particular, the fast upstrokeof the action potential is mediated by NaV channels.

The Vhalf of inactivation was -24 mV (n = 4), in good
agreement with the literature5,6.
BAVoltage (mV)NaV channels are in part characterized by their

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

-80

NaV1.8 is a TTXr channel. Compared with other NaVchannels, NaV1.8 has slow activation and inactivation

kinetics and is opened at relatively high voltages1. It is anpain, because modulation of NaV1.8 by inflammatory
mediators seems to be a key mechanism of DRG-200

200 pA

interesting drug target for inflammatory and neuropathic-40-100

10 msCImax (pA)

Different NaV channels play a key role in modulation

40-300

NaV1.8 has been reported to play an important role in theperception of cold pain .
3

In this Application Note we present data recorded on
the Patchliner® characterizing ND7-23 cells (a rat DRG/mouse neuroblastoma hybrid) stably transfected with rat
NaV1.8. All experiments were performed in the presenceof 100 nM TTX to block the endogenous TTXs Na+ current
present in these cells. The NaV1.8 activation and inactivation properties and tetracaine sensitivity recorded on

the Patchliner® were consistent with those reported in theliterature1-7.

Download more Application Notes from www.nanion.de-100-200-300

Imax (pA)

nociceptor sensitization and activation2. Interestingly,-400
Voltage (mV)
-80 -60 -40 -600

20Figure 1:
A Raw traces from an exemplar cell recorded on the Patchliner®. Shownare current responses to increasing voltage steps from -80 to +60 mV.
B Average current-voltage plot, Vhalf of activation was -9 mV (n = 19).
C Average inactivation plot, Vhalf of inactivation was -24 mV (n = 4).

Application Note
1.0

Figure 2 shows current responses to a single stepprotocol to 20 mV and inhibition of the NaV1.80.8Imax (nA)

200 pA

Control 10 ms
1 µM Tetracaine
10 µM
30 µM
100 µM
1 mM
Washout

tspan = 685 s

0.0-0.2-0.4

0.60.40.2
Vhold -90 mV
Vhold -120 mV-0.6
0.0-0.8
Control
1µM
10µM
30µM
100µM
1mM
Washout

B

A

1-(I/Imax)

current by increasing concentrations of tetracaine. The
timeplot of the experiment is also shown.

Tetracaine
Vhold -90 mV IC50 = 35 ± 8 µM (n = 3)
Vhold -120 mV IC50 = 74 ± 15 µM (n = 4)

Tetracaine

10 nM 100 nM 1 µM 10 µM 100 µM 1 mM
[Tetracaine]

Figure 3:
Average concentration response curve for tetracaine, IC50 = 35 ± 8 µM
(n = 3) for Vhold - 90 mV and 74 ± 15 µM (n = 4) for Vhold - 120 mV.

Figure 2:
A Raw traces from an exemplar cell recorded on the Patchliner® showing
inhibition of current by increasing concentrations of tetracaine. Shownare current responses to a single step protocol to 20 mV for 25 ms from aholding potential of -90 mV. Current amplitude was completely recovered
upon washout of tetracaine (red trace). B Timeplot of the experiment.

The potency of tetracaine was affected by holding

The corresponding concentration response curve is

In conclusion, NaV1.8 expressed in ND7-23 cells can beshown in Figure 3 revealing an IC50 for tetracaine ofpotential, becoming less potent with a more negative
holding potential, IC50 = 74 ± 15 µM (n = 4) with a holding
potential of -120 mV.

reliably recorded on the Patchliner® with activation and

35 ± 8 µM (n = 3) with a holding potential of -90 mV. This isinactivation properties, and tetracaine senstitivity, inin good agreement with the literature7.

good agreement to those reported in the literature5,6,7.

References

Cell culture
Cells were cultured and harvested according to Nan-

1. Sangameswaran et al., 1996. JBC. 271(11): 5953-5956

ion’s standard cell culture protocol.

2. Porreca et al., 1999. PNAS. 96: 7640-7644
3. Zimmermann et al., 2007. Nature. 447: 856-859
4. Goldin AL., 2001. Annu. Rev. Physiol. 63: 871-894
5. Catterall et al., 2005. Pharmacol. Rev. 57:397-409
6. Leffler et al., 2007. JPET. 320:354-364
7. John et al., 2004. Neuropharmacology. 46(3):425-38

Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for thePatchliner®. All recordings were made in the presence of
100 nM TTX to block endogenous TTXs NaV currents.

Methods

Current-voltage recordings were made using voltage

Cells

steps, from a holding potential of -120 mV. Inactivation

ND7-23 cells stably expressing rat NaV1.8 were suppliedfollowed by a step to 20 mV for 25 ms. Pharmacology

steps from -80 mV to 60 mV for 20 ms increasing in 10 mV

by Millipore.

protocol used a 100 ms pre-pulse to the voltage indicatedexperiments used a single voltage step protocol to 20 mV
for 25 ms from a holding potential of -90 mV or -120 mV(as indicated), repeated every 5 s.

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