Application Note

Channel:Cells:
Tools:

hNaV1.5
HEK293Patchliner®

Pharmacology of hNaV1.5 recorded onNanion´s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by EMD Millipore, USA.

Summary

Results

Voltage gated sodium channels (NaV) are important ele-Current responses of an individual cell to 20 ms voltage

ments of action potential initiation and propagation inpulses (-40 mV) in the presence of TTX concentrations as

excitable cells. The channels are activated upon a depo-indicated are shown in Figure 1. A single application oflarization of the membrane. Their activation leads to fur-external solution (Figure 1, washout) led to full recovery ofther depolarization of the membrane which constitutes

the peak control current.

the upstroke of the action potential.
NaV currents generally activate very fast (within 1-2 ms)
upon depolarization of the membrane. Hence, a goodand stable access resistance is critical for high quality
pharmacological patch clamp recordings. Also, for automated patch clamp devices, it is not a given that appliedcompound concentrations are accurately delivered tothe cell. This is a pre-requisite for accurately reproducible
dose-response curves.
Here we present data collected on the 8-channel Patchliner®. Tetrodotoxin and lidocaine dose‑response curves onhNaV1.5 expressed in HEK293 cells are shown. Lidocaine

control
0.3 µM
1 µM
3 µM
10 µM
30 µM
washout

has been shown to block hNaV1.5 in its inactivated state

10 ms(Bean et al. 1983) which means that the IC50 of lidocaine

becomes dependent on the holding potential. This dependence was investigated.
We also demonstrate the stability and reproducibility ofthe data collected with the Patchliner®. Using two sequential dose responses of hNaV1.5 to TTX we demonstrate that the compound concentrations are accurately
delivered to the cells and that recordings are stable withrobust access resistance.

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200 pA

Figure 1:
TTX dose-response curve on an individual cell.

Figure 2 shows the time-course of the peak Na+ current
during a single experiment. Two full TTX dose responses, including washout, were performed on a single cell. The reduction in peak currents at the different TTX concentrations

arehighlyreproducible

indicating

accuratecompound application. Washes were performed twice
after application of the highest TTX concentration. Full recovery of the peak current after the first wash is also obvious from the trace. Washing a second time did not lead toa significant change in peak current. This observation is
also an indication that solution changes are close to complete.
10I max in pA

030

wash

103-400

wash- 70 mV- 80 mV- 90 mV-100mV-110mV

0.80.60.40.2
0.0-110010123101010
concentration of lidocaine (µM)

410Figure 3:
The IC50 of lidocaine depends on the holding potential. Average IC50s of
1.7 mM (-70 mV), 4.0 mM (-80 mV), 21.7 mM (-90 mV), 37.8 mM (-100 mV), and194.6 mM (-110 mV) were obtained.

10.3

1.0

wash

3

1-20030

normalized peak current I Na

Application Note

Full lidocaine dose-response curves (in mM: 1, 3, 10, 30,0.3

100, 300, 1000, 3000) at all holding potentials (in mV: -70,

wash-80, -90, -100, -110) were obtained on all cells. The IC50-600of 195 mM at the holding potential of -110 mV is in good-800

agreement with 353 mM at -120 mV determined by Bean
04080

120160200240

280

Time in Seconds

et al. (1983).
In summary, whole cell recordings on the Patchliner®

Figure 2:
Time-course of the peak Na current in a single experiment. TTX was applied
and washed out as indicated by the arrows. Concentrations shown aregiven in mM.

are stable so that long lasting, high quality recordings
can be obtained. This, in combination with precise compound application, ensures reliable, reproducible doseresponse curves.

References

Electrophysiology

1. Bean, B.P., Cohen, C.S., and Tsien, R.W. 1983. Lidocaine

Whole cell patch clamp recordings were conducted

Block of Cardiac Sodium Channels. J.Gen. Physiol. 81:

according to Nanion’s standard procedure for the613 - 642.

Patchliner®. In the tetrodotoxin (TTX) experiments, currents
were elicited every 1 s by 20 ms voltage steps to -40 mV

Methods

from the holding potential of -90 mV. In the lidocaine
experiments, cells were held at the potentials as indicated.

Cells

Currents were elicited by 10 ms steps to 0 mV every 2 s.

PrecisION hNav1.5-HEK recombinant cell line (CYL3004)
were kindly supplied by EMD Millipore, USA.

Both TTX and lidocaine were diluted in external solution at
the indicated concentrations.

Cell culture
Cells were cultured and harvested according to Nanion‘sstandard cell culture protocol.

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