Application Note

Channel:Cells:
Tools:hASIC1a
CHO
SyncroPatch 384i

Pharmacology of human ASIC1a channels
on Nanion’s SyncroPatch 384i
Theelectrophysiology

team

at

NanionTechnologies

GmbH,

Munich.

Cells kindly provided by Charles River.

Summary

Results
proton-ASICs are activated by extracellular acidosis mediating

gated ion channels which are highly sensitive toa cation-conductance. Figure 1 shows success rate (Fig.

extracellularto1A), raw current trace (Fig. 1B inset) and pH-activation

cations1, predominantly Na+. They are members of the

response curve (Fig. 1B) for ASIC1a-mediated current

sodium-selective cation channels belonging to theactivation by solutions at different pH.
A 100 start of experimentpermeable

channel/degenerin(ENaC/DEG)

family2.

Wells (%)

To date, six different ASIC subunits (1a, 1b, 2a,2b, 3 and 4) encoded by four genes have been
identified . Three subunits assemble to form homomeric or
3channels.

They

are

found

expressed

other

is

contributelocalized
toinsynaptic

allcellulartransmission .
5neurological
areas

which

Further,

itisexpressed in various regions of the mammalian brain
includingthehippocampus,

cerebralcortex,cerebellum, and amygdala6-8. Therefore, ASIC1a is
involved in synaptic plasticity, learning and memory
and pathophysiology such as psychiatric dysfunction,
seizure and neuronal injury3,8,9.
Here we present high quality data at a high throughput

collected

activation
in

CHO

and
cells.

onthe

SyncroPatch

inhibition
The

pH

of
which

sa
bl

and

384i

ASIC1aelicitedshowing
expressedahalf-maximal response was in good agreement with theliterature3,6,10. The IC50s for block of the ASIC1a current by

amiloride and benzamil, known blockers of ASIC and ENaC
channels, were also in good agreement with theliterature2,6,11. Success rates of over 80% for completed
experiments were recorded.

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20

di

pain,

B

Normalized responseanddiseases such as ischaemia and inflammation4.

400throughout the CNS and PNS and have a proposed role innociception

60

edheteromeric

ASIC1aend of experiment

80

25

are

>1

sodium

and

are

25

acidosis(ASICs)-1channels

25

epithelial

ion

<2
5

Acid-sensing

Seal Resistance (MΩ)
EC50 = 367 nM = pH50 6.44
n = 378

2.5
2.0

pH 6.5
1.5
1.0
0.5

2 nA

0.5 s

0.0
3

pH 7.5

4

5

6

7 8 9

10-7

2

3

4

5

Concentration (M)pH 7.0pH 6.5

6

7 8 9

10-6

pH 6.0Figure 1: Activation of ASIC1a by decreasing pH. A. Success rate (sealresistance) of wells from one experiment recorded on a 4-hole NPC-384 chip.
Shown is a bar graph of seal resistances at the start (light blue) and endof the experiment (dark blue). B. pH response curve calculated across thewhole plate where currents were normalized to current elicited by pH 6.5 isshown. The curve was fitted with a Hill equation and the pH0.5 calculated to
be pH 6.44 ± 0.08 (n = 378), in excellent agreement with the literature3,6,10.
Raw trace of ASIC1a current activation in response to pH 6.5 solution is shownin the inset. Holding potential was -80 mV.

Application Note
In the experiment shown in Figure 1, currents were first

Amiloride is a potassium-sparing diuretic used to treat

activated with pH 6.5, to obtain a stable signal and to

hypertension and congestive heart failure. It is a known

check current reproducibility. Following this, the test pH

blocker of ASICs and ENaC, acting as a pore-blocker of

was applied and this was repeated twice with washout

ASICs6. Benzamil is an analog of amiloride with higher

between each addition. The responses were normalized

blocking potency. Figure 3 shows the concentration

to the current elicited by pH 6.5 and the pH response

response curve for amiloride and benzamil block of

curve calculated across the whole plate. The pH which

ASIC1α. ASIC1α-mediated currents were activated using

elicited the half-maximal current, pH0.5, was pH 6.44 ± 0.08

pH 6.5 solution and the concentration response curve for(n = 378), in excellent agreement with the literature range

each compound was calculated for each well, 4 con-of pH 6.4 - 6.63,6,10. The success rate for this experiment wascentrations of the compound were added to each well.

96.4 % determined at the end of experiment.

(see Figure 4). Amiloride and benzamil blocked ASIC1α

The reproducibility of ASIC1a-mediated currents isdemonstrated by applying pH 6.5 solution 7 times in thesame cell. Figure 2 shows raw current traces of a single

mediated currents with an IC50 of 6.0 ± 0.47 µM (n = 103)
and 1.36 ± 0.08 µM (n = 106), respectively, in excellent
agreement with the literature2,6,11).

A 1.0

well (Fig. 2A) and the average current amplitude over(Fig. 2B). ASIC1a mediated currents could be reliably
recorded on the SyncroPatch 384i.

A
pH 6.5

control
1 µM
3 µM
10 µM
30 µM

0.8
Normalized responsetime of 64 successfully recorded wells in one experiment

0.6

1 nA

0.5 s
0.4
0.2

Amiloride
IC50 = 6 µM ± 0.47 µM
n = 103

0.0
2 nA

B

10-7
1s

B

2

3

2

4 5 6

Normalized response

Imin (nA)

-4
-6
-8

4 5 6

1 nA

0.6

400

600

1000
800
Time (s)

1200

1400

1600

Figure 2: Reproducibility of ASIC1α-mediated currents recorded on the
SyncroPatch 384i. The experiment shows current activation in response
to 7 consecutive applications of acidified solution in order to evaluate
reproducibility of the signal. The experiment was performed on multi holechips (4x) and the holding potential was -80 mV. A. Raw current traces froman example well showing repetitive current activation by pH 6.5 solution. B.
Online analysis plot of one experiment showing average peak amplitude
versus time of 64 wells. The peak amplitudes were very similar with eachapplication of pH 6.5.

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4 5 6

10-4

0.5 s

0.4

BenzamilIC50 = 1.36 µM ± 0.08 µM
n = 106

-12
200

3

control
0.5 µM
1.5 µM
5 µM
15 µM

0.8

0.2

-10

2

1.0

0
-2

3

10-6
10-5
Concentration (M)

0.0
10-7

2

3

4 5 6

2

3

4 56

10-6
10-5
Concentration (M)2

3 4 5 6

10-4

Figure 3: Average concentration response curve for amiloride and benzamilblock of ASIC1α. The concentration response curve was constructed fromcumulative additions of increasing concentrations of compound in individual
wells as shown in Figure 4. The SyncroPatch 384 analysis software (DataControl384) was used to calculate the average concentration response curve,
normalized to maximum activation and fitted with a standard Hill-equation.
A. The IC50 for block of ASIC1α with amiloride was 6.0 ± 0.47 µM (n = 103), andB, with benzamil 1.36 µM ± 0.08 µM (n = 106), both in excellent agreementwith the literature2,6,11.

Application Note

Figure 4: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of online analysis of
ASIC1a-expressing CHO cells as recorded on one NPC-384 (multihole, 4x) patch clamp chip. Positive Control (pH 7.4), amiloride, benzamil or negative
control (0.3 % DMSO) in increasing concentrations was applied to 4 columns for each compound . 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 > 130 MΩ), blue (Rmemb = 25 - 130MΩ), light blue or grey (Rmemb < 25 MΩ or cells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayedon the right. Graphs show current amplitudes over time of ASIC1a channels following activation by pH 6.5 for three times (grey region) and inhibition bycompound at four increasing concentrations indicated (increasing shades of blue regions).

Exp end and chip
unload5 min

30 - 35 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 3 control applications (pH 6.5)
plus 4 compound additions and 1 wash between each
addition

Analysis

Figure 5: Time line of an experiment on the SyncroPatch 384. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a
4 point cumulative concentration response curve on ASIC1a-mediated currents including 3 reference additions and one wash between each addition
took approximately 35-40 min.

Download more Application Notes from www.nanion.deApplication Note
Figure 4 shows a screenshot of the SyncroPatch 384

activation

software during an experiment. A color-coded over-consecutive

view (based on seal resistance in this case) of all

wash with buffer between each addition (Figure 5).

384 wells gives the user a good impression of the

The IC50 for amiloride and benzamil block of ASIC1α-success rate of the experiment. The user can easilyofcurrents

inconcentrations

theofpresence
blocker

of

and

4

one

mediated currents calculated using the SyncroPatch

toggle between raw traces and online analysis. In the

384’s

example shown, online analysis is chosen and the graphs

excellent agreement with the literature2,6,11.

analysis

software,

DataControl

384,

was

in

show control responses to pH 6.5 (grey region) and current
responses in the presence of amiloride or benzamil at

The SyncroPatch 384i is a high throughput and highly

increasing concentrations (increasing shades of blue re-reliable automated patch clamp device for recordinggion). An individual well can be highlighted to monitor

ASIC1α currents. User-friendly software, excellent successprogression of the experiment and is shown enlarged at

rates, single additions or multiple additions of compound

the bottom of the screen.

to each well and easy analysis result in reliable highquality data at an increased throughput with an

In conclusion, ASIC1α expressed in CHO cells can be

economical cost per data point.

recorded on the SyncroPatch 384 with high successrates for completed experiments (typically >80%). The
time line of each experiment was about 35 - 40 minutes(start – end) and included 3 control applications withpH solution followed by pre-incubation with blocker andReferences
1. Lingueglia, E. & Lazdunski, M. 2013. WIREs Membr.
Transp. Signal. 2:155–171
2. Kellenberger, S. & Schild, L. 2002. Physiol. Rev. 82: 735767
3. Wemmie, J.A., et al., 2013. Nat. Rev. Neurosci. 14(7):461–471
5. Sluka, K.A., et al., 2009. Curr. Opin. Drug Discov. Devel.
12(5): 693–704
6. Wemmie, J.A., et al., 2003. J. Neurosci. 23, 5496–5502
7. García-Añoveros, et al., 1997. Proc. Natl. Acad. Sci.
U.S.A. 94, 1459–1464
8. Waldmann, R. et al.,1997. Nature 386, 173–177
9. Wemmie, J. A. et al., 2002. Neuron 34, 463–477
10. Gunthorpe M.J., et al., 2001. Pflugers Arch. 442(5):66874.
11. Leng, TD., et al., 2016. CNS Neurosci Ther. 22(6):468476.

Cell culture
Cells were cultured and harvested according toNanion’s standard cell culture protocol. Channel expression was induced by incubation in 1 μg/ml tetracycline
3-6 hours prior to experiments.
Electrophysiology
Perforated patch clamp recordings were conducted on
the SyncroPatch 384i or the predecessor model, theSyncroPatch 384PE, according to Nanion’s standard
procedure using an internal solution containing 25 µM
escin. Cells were held at a constant holding potential of-80 mV. Cells were activated 3 times with pH 6.5 tomonitor reproducibility of the signal and to elicit maximum
peak amplitude. Compounds were pre-incubated in pH
7.4 for 3 minutes. Responses after compound addition
were normalized to the response evoked by pH 6.5.
Cumulative compound concentration curves were

Methods

constructed and fitted with Hill equation.

Cells

hASIC1α expressing CHO cells are kindly provided by
Charles River.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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