CYL3025-MM04 hNav1.8.pdf - Millipore

PrecisION hNav1.8/β1-HEK 

Recombinant Cell Line 

Catalog Number: CYL3025 

PRODUCT DESCRIPTION: 

Recombinant HEK293 cell line expressing the human Nav1.8 (accession number AF117907; tetrodotoxin-resistant voltage-gated 

sodium channel type X (SCN10A)) and the human sodium channel beta 1 subunit (accession number NM_001037; SCN1B). 

ASSOCIATED PRODUCTS: 

The PrecisION hNav1.8/β1-HEK Recombinant Cell Line is provided to customers on the purchase of an appropriate 

license. The available licenses are: 

CYL3025SS PrecisION hNav1.8/β1-HEK Single Site License 

CYL3025TS PrecisION hNav1.8/β1-HEK Two Site License 

CYL3025MS PrecisION hNav1.8/β1-HEK Multiple Site License 

CONTENTS: 

2 x 1 mL aliquots containing 1.2 x 10 6 cells/mL in 10% DMSO at passage 17. 

STORAGE: 

Vials are to be stored in liquid N2. 

WARNINGS: 

For Research Use Only; Not for Use in Diagnostic Procedures 

Not for Animal or Human Consumption 

GMO 

This product contains genetically modified organisms. 

Este producto contiene organismos genéticamente modificados. 

Questo prodotto contiene degli organismi geneticamente modificati. 

Dieses Produkt enthält genetisch modifizierte Organismen. 

Ce produit contient organismes génétiquement des modifiés. 

Dit product bevat genetisch gewijzigde organismen. 

Tämä tuote sisältää geneettisesti muutettuja organismeja. 

Denna produkt innehåller genetiskt ändrade organismer. 

MYCOPLASMA TESTING: 

The cell line has been screened using the ELISA based Mycoplasma Detection kit (Roche) and by a PCR VenorGem kit (Minerva 

Biolabs) to confirm the absence of Mycoplasma species. 

FUNCTIONAL VALIDATION: 

The electrophysiological and pharmacological properties of the cell line were examined using conventional whole-cell recording 

techniques. The current voltage relationship and activation/inactivation kinetics were similar to previously reported data for 

hNav1.8/β1 currents (Vijayaragavan et al., 2001; 2003; Vijayaragavan et al., 2004)). The currents were resistant (insensitive) to block 

by 10 µM TTX. The current were also blocked by tetracaine, lidocaine and lamotrigine. Functional channel expression over time was 

monitored using PatchXpress. At least 75% of cells expressed hNav1.8/β1 currents over 500 pA for 30 passages. The mean 

current was 900 ± 17 pA (n = 11). 

RECOMMENDED CULTURE CONDITIONS: 

Recommended culture conditions and standard operating procedure are provided with the product. 

MM04-DJ-CYL3025 

DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery




FUNCTIONAL VALIDATION: 

Introduction. 

The voltage-gated sodium channel α-subunit Nav1.8 (also known as hSCN10A, SNS and PN3) is expressed in high levels in 

mammalian adult dorsal root ganglion (DRG) neurons where it is involved in action potential initiation and transmission of nociceptive 

impulses (Leffler et al., 2007). Native sodium channels are multi-subunit complexes, composed of not only the pore forming α 

subunit, but also auxiliary β subunits. The β1 subunit is known to increase peak current amplitude as well as increasing sodium ion 

channel expression at the cell surface (Vijayaragavan et al., 2004). 

Electrophysiological Properties of the hNav1.8/β1 current. 

Whole-cell Patch Clamp Electrophysiology. 

Current/Voltage Relationship: 

Activation: Activation of the hNav1.8/β1 produced an inward current with slow inactivation kinetics (Figure 1). The channel 

activated at approximately -40 mV and reached its peak current at 20 mV (Figure 2). The hNav1.8/β1 channel had a half-activation 

voltage (Vi) of -1.2 ± 0.7 mV and a slope (k) of 11.4 ± 0.6 (Figure 3). This is close to previously reported values (V½ = -3 to 2.5 mV 

and k = 5.5 – 11.3; Rabert et al., 1998; Vijayaragavan et al., 2001; 2003; Vijayaragavan et al., 2004). 

Figure 1. Typical current traces for hNav1.8/β1 evoked by the depolarizing voltage steps. 

The currents were produced by 100 ms voltage steps from –90 to +60 mV in 10 mV increments. Sweep every 10 s. 

The cells were held at a holding potential of –90 mV. The voltage protocol used is shown in red. 


0.2 nA 

50 mV 

DDServices@merckgroup.com ● www.millipore.com/DrugDiscovery 

5 ms 

5 ms




Figure 2. Typical Current-voltage (I/V) relationship for hNav1.8/β1. 

Figure 3. Conductance-voltage graph of the hNav1.8/β1 current. 

The conductance was normalized to peak conductance at +20 mV and plotted against membrane voltage. The 

voltage protocol used is shown (inset). 


-120 mV 


20 mV 

5 ms




Inactivation: 

The hNav1.8/β1 channel had a half-inactivation voltage (Vi) of -47.6 ± 0.9 mV and a slope (k) of 9.5 ± 0.8 (Figure 

4). Again this is close to values reported previously (V½ = -40 to –63 mV and k = 7.1 – 12.9; Vijayaragavan et al., 

2001; Vijayaragavan et al., 2004; Leffler et al., 2007). 

Figure 4. Steady-state inactivation of the hNav1.8/β1. 

The voltage protocol used is shown (inset). 


20 mV 

1 s 





Recovery from inactivation: 

In order to assess the time constant of recovery of inactivation at -110 mV following an inactivating 20 ms voltage 

step to 10 mV, a double pulse protocol was used. First, the membrane was stepped from the holding potential (- 

110 mV) to 10 mV for 20 ms (Pulse 1, P1). The holding potential of -110 mV was sufficient to remove any 

inactivation so that the subsequent test pulse to 10 mV produced maximal inward current (i.e. all channels were 

available for activation). Immediately after the step to 10 mV the voltage was returned to -110 mV for varying 

amounts of time ranging from 1 – 38.4 ms with each successive intervals increasing by 50% (interpulse duration). 

After each incremental change in time, the voltage was stepped back to 10 mV (Pulse 2, P2). This was to assess 

the amount of recovery from inactivation at each incremental time point at -110 mV, by comparing the amplitude of 

P2 (after returning to -110 mV) with P1. Clearly, if channels had fully recovered at a given incremental time point, 

then the amplitude of P2 would be the same as P1, hence P2/P1 = 1. However, if channels had only partially 

recovered at a given incremental time point, the amplitude of P2 would be smaller, so that P2/P1 =




Pharmacological Properties of the hNav1.8/β1 current. 

Pharmacology - Tetrodotoxin: 

The puffer fish derived toxin, tetrodotoxin, has been found to block multiple sodium channels in the nanomolar 

range. The hNav1.8 channel is very resistant to block by TTX, with an IC50 of 60 - 80 µM (Akopian et al., 1996; 

Rabert et al., 1998). In order to conclusively demonstrate the functional expression of hNav1.8 currents, 10 µM 

TTX was tested on the inward currents evoked by stepping to a test potential of 20 mV from a holding potential of - 

90 mV (Figure 6). Currents following the addition of 10 µM TTX were 93.4 ± 1.8% (n = 11) of those preceding the 

addition of TTX. 

Figure 6. The effect of 10 µM tetrodotoxin on hNav1.8/β1 currents. 

Typical traces showing the effect of 10 µM tetrodotoxin. Sodium currents are shown before and in the presence of 

tetrodotoxin. Black trace represents inward current before addition of TTX and the red trace the current in the 

presence of TTX. The cells were held at a holding potential of –90 mV and then stepped to a test pulse to 20 mV 

for 20 ms at a frequency of 0.1 Hz. 


TTX 10 µM 

250 pA 


200 

5 

20 s




Pharmacology – A-803467: 

A-803467 is a sodium channel blocker that potently blocks hNav1.8 with an IC50 in the nanomolar range whilst 

blocking other Nav channels in the micromolar range (Jarvis et al., 2007). hNav1.8 currents evoked by repetitive 

(10 Hz) 20 ms depolarizing pulses from a holding current of either –90 mV or –60 mV were blocked by 100 nM by 

42-43% at -90 mV and by 62-70% at -60 mV (Figure 7). 

Figure 7. The block of hNav1.8/β1 by 100 nM A-803467 

The upper panel shows peak inward current plotted for each consecutive pulse, before, in the presence of 

A803467 (red bar) and after wash off of drug using a Vhold of –90 mV (left) or –60 mV (right). The lower panel 

shows representative individual current traces at the indicated time points. 






Pharmacology – Tetracaine, Lidocaine and Lamotrigine: 

The potency of the block of the hNav1.8/β1 by tetracaine, lidocaine and lamotrigine when 50% of the channels are 

in the inactive state cells was assessed. Cells were voltage clamped at a holding potential of -90 mV then stepped 

to -50 mV (~ V½ inactivation) for 5 seconds followed by a test pulse to 20 mV. This was repeated every 10 

seconds. hNav1.8/β1 was blocked with IC50 values of 0.6 ± 0.2 µM (n = 9), 63.3 ± 12.2 µM (n = 6) and 202.3 ± 22.5 

µM (n = 6) for tetracaine, lidocaine and lamotrigine respectively (Figure 8). These values are in agreement with 

published findings (Poyraz et al., 2003; Jarvis et al., 2007). 

Figure 8. Block of hNav1.8/β1 current using tetracaine, lidocaine and lamotrigine. 

A. Dose-response curves of tetracaine, lidocaine and lamotrigine on the hNav1.8/β1 current. Voltage protocol 

shown in inset. 

B. Typical current traces showing the inhibition of hNav1.8 currents by increasing doses of lamotrigine. Control 

current before lamotrigine addition shown in black. 

A 

B 


1000 µM 

100 µM 

10 µM 

1 µM 

0.2 nA 

5 ms 





Stability of hNav1.8/β1–HEK293 Cell Line. 

PatchXpress Electrophysiology. 

The hNav1.8/β1–HEK293 cell line has stable expression for >30 passages. 

Figure 8. Stability of expression over passage. 

The upper panel shows the percentage of cells expressing a mean peak inward current >500 pA for cell passages 

9, 14, 19, 26. The numbers above each bar represent the number of cells sealed (out of a maximum of 16). 






Vectors: 


AhdI 

PvuI 

Amp 

PCIN5_NEWC 

4904 bps 

Polylinker: CMV-BamHI-NotI-hNav1.8-NotI-AscI-NdeI-EcoRI-IRES-neo 

4000 

3000 

HincII 

XhoI 

AvaI 

SphI 

HincII 

SspI 

hNav1.8‐pCIN5 

polyA 

XbaI 

BclI 


2000 

MluI 

HincII 

CMV promoter 

Neo 

1000 

BssHII 

SphI 

NdeI 

SnaBI 

IRES 

PstI 

BamHI 

ApaI 

NotI 

AvaI 

XhoI 

AscI 

BssHII 

NdeI 

ClaI 

HpaI 

HincII 

EcoRI




Polylinker: CMV-KpnI-SacI-BamHI-hNav β1-NotI-IRES-hyg 

hNav1.8 Sequence (AF117907) 

hNav β1 Sequence (NM_001037) 


PvuII 

XhoI 

PvuII 

SphI 

4000 

Amp 

BclI 

SacI 

XbaI 

5000 

polyA 

SspI 

pCIH 

PCIH.SEQ 

5726 bps 


3000 

CMV promoter 

hNav β1‐pCIH 

Hygro 

1000 

2000 

NdeI 

IVS 

IRES 

PstI 

NdeI 

SacI 

KpnI 

SacI 

BamHI 

BstXI 

PstI 

SphI 

NotI 

ApaI 

KpnI




References 

Akiba, I., Seki, T., Mori, M., Iizuka, M., Nishimura, S., Sasaki, S., Imoto, K. and Barsoumian, E.L. (2003). Stable 

expression and characterization of human PN1 and PN3 sodium channels. Receptors Channels 9, 291-299. 

Akopian, A.N., Sivilotti, L. and Wood, J.N. (1996). A tetrodotoxin-resistant voltage-gated sodium channel 

expressed by sensory neurons. Nature 379, 257-262. 

Jarvis, M.F., Honore, P., Shieh, C.C., Chapman, M., Joshi, S., Zhang, X.F., Kort, M., Carroll, W., Marron, B., 

Atkinson, R., Thomas, J., Liu, D., Krambis, M., Liu, Y., McGaraughty, S., Chu, K., Roeloffs, R., Zhong, C., Mikusa, 

J.P., Hernandez, G., Gauvin, D., Wade, C., Zhu, C., Pai, M., Scanio, M., Shi, L., Drizin, I., Gregg, R., Matulenko, 

M., Hakeem, A., Gross, M., Johnson, M., Marsh, K., Wagoner, P.K., Sullivan, J.P., Faltynek, C.R. and Krafte, D.S. 

(2007). A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and 

inflammatory pain in the rat. Proc Natl Acad Sci U S A 104, 8520-8525. 

Leffler, A., Reiprich, A., Mohapatra, D.P. and Nau, C. (2007). Use-dependent block by lidocaine but not 

amitriptyline is more pronounced in tetrodotoxin (TTX)-Resistant Nav1.8 than in TTX-sensitive Na+ channels. J 

Pharmacol Exp Ther 320, 354-364. 

Poyraz, D., Brau, M.E., Wotka, F., Puhlmann, B., Scholz, A.M., Hempelmann, G., Kox, W.J. and Spies, C.D. 

(2003). Lidocaine and octanol have different modes of action at tetrodotoxin-resistant Na(+) channels of peripheral 

nerves. Anesth Analg 97, 1317-1324. 

Rabert, D.K., Koch, B.D., Ilnicka, M., Obernolte, R.A., Naylor, S.L., Herman, R.C., Eglen, R.M., Hunter, J.C. and 

Sangameswaran, L. (1998). A tetrodotoxin-resistant voltage-gated sodium channel from human dorsal root 

ganglia, hPN3/SCN10A. Pain 78, 107-114. 

Vijayaragavan, K., O'Leary, M.E. and Chahine, M. (2001). Gating properties of Na(v)1.7 and Na(v)1.8 peripheral 

nerve sodium channels. J Neurosci 21, 7909-7918. 

Vijayaragavan, K., Powell, A.J., Kinghorn, I.J. and Chahine, M. (2004). Role of auxiliary beta1-, beta2-, and beta3subunits 

and their interaction with Na(v)1.8 voltage-gated sodium channel. Biochem Biophys Res Commun 319, 

531-540. 


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©2009: Millipore Corporation. All rights reserved. No part of these works may be reproduced in any form without 

permission in writing. 

PrecisION is a trademark of Merck KG&A, 

Darmstadt, Germany.. IonWorks HT is a 

trademark of Molecular Devices Corporation.

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