Application Note

Channel:Cells:
Tools:hASIC3
HEK293
SyncroPatch 384PE

Activation and Inhibition of human ASIC3
Channels on Nanion’s SyncroPatch 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by EMD Millipore.

Results

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

For the evaluation of the performance of HEK-ASIC3

channels activated by protons. They are members ofcells, Seal Resistance, Cslow and Series Resistancethe sodium-selective cation channels belonging to the(Rseries) were determined from one experiment (Fig. 1) .
A 100
start of experimentendof experiment80

So far, 6 different ASIC subunits have been identified
encoded by 4 genes3. They are found expressed throughout the CNS and PNS and have a proposed role inagreement with a role in pain perception4. Furthermore,
ASIC3 is expressed in higher amounts in nociceptive
neurons innervating muscle (~ 50%) compared to skin(~ 10%), which indicates that ASIC3 may play animportant role in detecting muscle acidosis4.

sabl302010put collected on the SyncroPatch 384PE showing
activationinHEKandcells.

inhibition
ThepHofASIC3

which

elicited

expressed
ahalf-maximal response was in good agreement with theliterature3,6. The IC50 for block of the ASIC3 current
by amiloride, a known blocker of ASIC and ENaC

channels, was also in good agreement with theliterature2,6,7. Success rates of over 80% for completed
experiments were recorded.

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Here we present high quality data at a high through-Seal Resistance
60C50403020100010BCells (%)

fibers innervating the skin, muscles, joints and viscera, indifound to be localized to primary afferent nociceptiveM

channel was first identified in the late 1990’s . It was
500

0diseases such as ischaemia and inflammation4. The ASIC3Ω

20

neurological>5

otherCslow (pF)<551
10 0-1
15 5-2
20 0-2
5>2
5and

Ω

pain,

M

and40

Cells (%)

nociception

6000

and are permeable to cations2, predominantly Na+.

-5family1. ASICs are highly sensitive to extracellular acidosisΩ(ENaC/DEG)

M

channel/degenerin<100sodiumed

epithelial

Cells (%)

SummaryRseries (MΩ)

Figure 1: Statistics of ASIC3 expressing HEK293 cells recorded on one
NPC-384 chip on the SyncroPatch 384PE. Success rate (seal resistance) ofindividual HEK cells on the SyncroPatch® 384. Shown is a bar graph of seal
resistances at the start (light blue) and end of the experiment (dark blue).
B Bar graph of cell capacitance (Cslow) and C Series Resistance (Rseries)
values for HEK293 cells expressing ASIC3.

Application Note
Currents

mediatedbyASIC3

could

bereliably

recorded on the SyncroPatch 384PE with a highsuccess rate. Figure 2 shows a screenshot of the dataacquisition and analysis software of the SyncroPatch®
384PE during an experiment recording the current
response to 3 applications of pH 6.0 solution. In thisexperiment, 76.8% of cells had a seal resistance > 500 MΩ
with a further 8.1% with a seal resistance > 100 MΩ.

Figure 3: Activation of ASIC3 by decreasing pH. Currents were normalised
to the maximum peak amplitude elicited by pH 5.0 and the pH responsecurve calculated across the whole plate. The curve was fitted with a Hillequation and the pH0.5 calculated to be pH 6.45 ± 0.08 (n = 341), in excellent
agreement with the literature3,6.

Amiloride is a potassium-sparing diuretic used to treat
hypertension and congestive heart failure. It is a known
Figure 2: Typical recording from ASIC3 expressed in HEK cells on theSyncroPatch 384PE. The screenshot shows the data acquisition and analysis
software used on the SyncroPatch 384PE. Three hundred and eighty-four
small color-coded pictures shown in the upper left part from A1 to P24
display 384 recordings. Wells are color-coded based on seal resistance(green: Rmemb > 500 MΩ, blue: Rmemb = 100 - 500 MΩ, light blue or grey:
Rmemb < 100 MΩ or disabled). One highlighted experiment is displayed at
the bottom, 16 selected experiments are displayed on the right. On the farright, the online analysis view showing peak current amplitude is provided.
Vertical lines and blue shaded area show application of low pH solution
and white shows incubation in wash solution. The experiment shows current
activation in response to acidified solution at pH 6.0. This step was repeated
3 times (with wash between applications) to show reproducibility of thesignal. Holding potential was -60 mV.

blocker of ASICs and ENaC, acting as a pore-blocker of
ASICs6 . Figure 4 shows the concentration response curve
for amiloride block of ASIC3. ASIC3-mediated currents
were activated using pH 6.0 solution and the concentration response curve was calculated across the wholeplate (as shown in Figure 5). The IC50 for amiloride block ofASIC3 was 10.4 ± 0.1 µM (n = 326) in excellent agreementwith the literature (16 µM2; 5 - 100 µM6; 60 µM7).

ASIC3 could be reliably and reproducibly recordedon the SyncroPatch 384PE as shown in Figure 2. The
current responses to multiple applications of pH 6.0
solution could be exactly overlaid.
Figure 3 shows the response curve to decreasing pH. Cells
were activated by pH 5.0 to obtain maximum peak amplitude and to check reproducibility of the signal. Following this, the test pH was applied and this was repeated
3 times with washout between each addition. The responses were normalised to maximum current elicited by
pH 5.0 and the pH response curve calculated across thewhole plate. The pH which elicited the half-maximal current, pH0.5, was pH 6.45 ± 0.08 (n = 341), in excellent agreement with the literature value of pH 6.4 - 6.63,6.

Figure 4: Average concentration response curve for amiloride block of ASIC3.
The concentration response curve was constructed across the whole plate
as shown in Figure 5. The SyncroPatch 384PE analysis software (DataControl384) was used to calculate the average concentration response curve,
normalized to maximum block and fitted with a standard Hill-equation. TheIC50 for amiloride block of ASIC3 was 10.4 ± 0.1 µM (n = 326), in excellent
agreement with the literature2,6,7.

Nanion Technologies GmbHphone + 49 89 219 095 0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot of depiction of raw data tracesof ASIC3-expressing HEK cells as recorded on one NPC-384 patch clamp chip. A single compound (in this case amiloride) was applied to individual wells
at varying concentrations (including control wells) across the plate. The data of the 384 well plate representation in the upper left part are color-coded
for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb > 500 MΩ), blue (Rmemb = 100 – 500 MΩ), light blue or grey
(Rmemb < 100 MΩ or cells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs
show raw data traces of ASIC3 channels following activation by pH 6.0 (blue trace) and inhibition by amiloride at the concentrations indicated (blacktrace).

Exp end and chipunload
10 - 12 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 3 control applications (pH 6.0)
plus 1 - 2 compound additions

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch 384PE. The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) fora single point concentration response curve on ASIC3-mediated currents took approximately 17-19 min.

Nanion Technologies GmbHphone + 49 89 219 095 0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application NoteFigure 5 shows a screenshot of the SyncroPatch 384

on the SyncroPatch 384PE is in good agreement withsoftware during an experiment. A color-coded over-the literature3,6. The IC50 for amiloride block of ASIC3-view (based on seal resistance in this case) of all

mediated currents calculated using the SyncroPatch

384 wells gives the user a good impression of the384PE’s analysis software, DataControl 384, was insuccess rate of the experiment. The user can easily

excellent agreement with the literature2,6,7.

toggle between raw traces and online analysis. In theexample shown, raw traces are chosen and the graphs

The SyncroPatch 384PE is a high throughput and highly

show control responses to pH 6.0 (blue) and current

reliable automated patch clamp device for recording

responses

theASIC3 currents. User-friendly software, excellent successconcentrations indicatd (black). An individual well canin

thepresence

ofamiloride

atrates, single additions or multiple additions of compound

be highlighted to monitor progression of the experimentto each cell and easy analysis result in reliable highand is shown enlarged at the bottom of the screen.

quality data at an increased throughput with an
economical cost per data point.

In conclusion, ASIC3 expressed in HEK cells can berecorded on the SyncroPatch 384PE with high successrates for completed experiments (typically >80%). The
timeline of each experiment was about 17-19 minutes(start – end) and included 3 control applications withpH solution followed by pre-incubation with blocker andactivation of currents in the presence of 1 concentrationof blocker. The pH response curve for ASIC3 recorded

Cell culture

References
1. Kellenberger, S. & Schild, L. 2002. Physiol. Rev. 82: 735767
2. Lingueglia, E. & Lazdunski, M. 2013. WIREs Membr.
Transp. Signal. 2:155–171
3. Wemmie, J.A., et al., 2013. Nat. Rev. Neurosci. 14(7):461–471
4. Sluka, K.A., et al., 2009. Curr. Opin. Drug Discov. Devel.
12(5): 693–704
5. Waldmann, R., et al., 1997. J. Biol. Chem. 272(34):20975–20978
6. Deval, E., et al., 2010. Pharmacology & Therapeutics
128: 549–558
7. Chu, X-P., et al., 2011. Int J. Physiol. Pathophysiol. Pharmacol. 3(4):288-309

Cells were cultured and harvested according toNanion’s standard cell culture protocol.
Electrophysiology
Perforated patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch 384PE using an internal solution containing
25 µM Escin. Cells were held at -60 mV for the duration ofthe experiment. To calculate the pH response curve, cells
were activated 3 times with pH 5.0 to monitor
reproducibility of the signal and to elicit maximum peak
amplitude.

ThreetestpHsolutions

wereapplied

subsequently to each cell with washout betweenadditions. The responses were normalised to the maximum
response (pH 5.0) and the pH response curve calculated

Methods

across the whole plate. Amiloride was pre-incubated in
pH 8.0 solution for 150 s prior to co-application at pH 6.0.

Cells

HEK293 cells expressing hASIC3.

Nanion Technologies GmbHphone + 49 89 219 095 0

Ganghoferstr. 70A

fax80339 Munich, Germany

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