Application Note

Channel:Cells:
Tools:hASIC3
HEK293Patchliner®

Characterization of hASIC3 (HEK) onNanion´s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Millipore.

SummaryResults

Acid-sensing ion channels (ASICs) are ligand-gated ion

Current responses of an individual cell expressing

channels activated by protons and are members of

acid-sensing ion channel hASIC3 exposed to acidifying

the sodium-selective cation channels belonging to theexternal solution are shown in Figure 1. Short application

epithelial(ENaC/DEG)of acidified external solution (pH 6.8 – 5.5) resulted in sus-family. They are highly sensitive to extracellular acidosis.

tained increasing inward currents. Acidosis from pH 6.8

1

sodiumchannel/degenerin

to pH 5.5 leads to over 2-fold increase of inward currents
Inrodents,

where

they

aremainlyexpressedin(Fig. 1B).

neurons of the peripheral nervous system, ASIC3 plays
is correlated to tissue acidosis. However, the role of theA acidosis

human ASIC3 channel has not yet been elucidated.
sustained window current upon external acidification.
Here we present data recorded on an 8-channel
Patchliner. Current responses upon external acidification
as well as amiloride block of acidosis-induced currents of1 nA

In contrast to other ASIC ion channels, ASIC3 shows a500 ms

B
pH 6.8pH 6.1
pH 5.8pH 5.5Current density (nA/pF)

an important role as sensor of non-adaptive pain which

0.0-0.1

pH 6.8n=8pH 5.5
n=8-0.2-0.3-0.4-0.5

hASIC3-expressing HEK293 cells are shown.
Figure 1:
A Current responses of an individual cell expressing ASIC3 held at-70 mV upon wash in of solution with decreasing pH as indicated.
B Acidification from pH 6.8 to pH 5.5 leads to over a 2-fold increase ininward currents in hASIC3-expressing HEK293 cells.

Download more Application Notes from www.nanion.deApplication Noteamiloride on hASIC3 expressing HEK293 cells is shown.
Inward currents induced by acidic external solution(pH 5.5) are suppressed by application of 1 µM – 1 mM
amiloride revealing an IC50 of 26.0 ± 3.3 µM (n = 14) asshown in Figure 3, in good agreement with values reported
in the literature1-8.

1 nAControl1 µM Amiloride
5 µM Amiloride
10 µM Amiloride

500 ms

50 µM Amiloride
0.1 mM Amiloride
0.5 mM Amiloride
1 mM Amiloride

Normalized Ipeak

In Figure 2 the effect of the known DEG/ENaC inhibitor
0.80.60.4
0.2
0.0
10 nM 100 nM 1 µM

10 µM 100 µM 1 mM

[Amiloride]

Figure 3:
Concentration response curve for amiloride of hASIC3-expressing HEK293
cells collected on the Patchliner. IC50 = 26.0 ± 3.3 µM (n = 14).

In summary hASIC3 ion channels stably expressed inHEK293 cells show reliable acidosis-induced inward
currents that can be blocked by amiloride.

Figure 2:
A Current responses of an individual cell expressing ASIC3 to pH 5.5 solution(holding potential -70 mV) and block by increasing concentrations ofamiloride (concentrations as indicated).

Therefore, the Patchliner provides a viable, higher
throughput alternative to conventional patch clamp forinvestigation of active hASIC3 lead compounds.

Cell culture

References

Cells were cultured and harvested according to
1. Waldmann, et al., 1997. Nature. 386 (6621): 173-177

Nanion’s standard cell culture protocol.

2. de Weille et al., 1998. FEBS Letters. 433 (3): 257-60
3. Chen et al.,1998. PNAS. 95 (17): 10240 – 10245
4. Benson et al., 1999. Circ. Res. 84 (8): 921-928
5. Chu et al., 2002. J. Neurophysiol. 87 (5): 2555-2561
6. Wu et al., 2004. J. Biol. Chem. 279 (42): 43716–43724
7. Yermolaieva et al., 2004. PNAS. 101 (17): 6752-6757
8. Jiang et al., 2009. Neuroscience. 162 (1): 55 – 66

Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for thePatchliner. Cells were held at a holding potential of-70 mV. To achieve short exposure times, solutions werestacked in the robotic pipettor. First, 80 µl wash solution

Methods

was aspirated followed by 40 µl of the agonist-containing

Cells

20 µl/s.

solution and then applied to the cell at a speed of

HEK293 cells stably expressing hASIC3 were supplied byMillipore.

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