Development and validation of ASIC1a ligand-gated ion channel drug discovery
assays on automated patch clamp platforms.
Marc Rogers1, Sam Manyweathers1, Tom A. Goetze2, Nadine Becker2, Ilka Rinke-Weiß2, Michael George2, Alison Obergrussberger2
1Metrion Biosciences, Cambridge, UK; 2Nanion Technologies, Munich, Germany● www.nanion.de ● info@nanion.de ●• Records both time course and voltage
Introduction
dependence of compound application

High throughput activation of ASIC1a

ASIC1a conductance trackingAA

Calculation of crude
Vhalfis possible
for eachAcid-sensing ion channels (ASICs) are• proton-gatedionchannels
which
aresweephighly sensitive to extracellular acidosis and are permeable to cations1,• Tau of activation is also available for different+
predominantly Na . To date, six different voltages
ASIC subunits(1a,1b,2a, 2b, 3 and 4)
throughout
therecording
encoded by four genes have been identified2. Three subunits assemble toform homomeric or heteromeric channels which are expressed throughout theCNS and PNS. Excitatory ASIC channels have a proposed role in nociception
and pain, and other neurological diseases such as ischaemia, inflammationand learning and memory2.

Δ𝑉𝑐Δ𝑉𝑑Ligand-gated ion channels with rapid activation and desensitization present
specific challenges on automated patch clamp (APC) systems due to low
channel expression, the need for rapid application and wash-off of ligands,
and loss of responsiveness due to short- and long-term desensitization. In thisstudy we used ASIC1a channels expressed in CHO cells as an exemplar rapid
ligand-gated ion channel to develop reliable screening assays on medium(Patchliner) and high throughput (SyncroPatch 384i) APC devices. ASIC1amediated current responses were reproducible and could be repetitively
activated with similar peak amplitudes at least 7 times in the same cell using
techniques which minimize agonist exposure time. ASIC1a was activated bydecreasing pH and blocked by benzamil and amiloride. Finally, wedemonstrate successful implementation of the classic historical input
resistance/conductance-tracking technique to measure ligand-gated current
activation on an APC platform.

HCN2 Pharmacology in High Throughput

BCCHO•

Agonists increase membrane conductance and reduce cell resistance•Small current injections are used to track conductance changes from Vm

B. Control

D.

ASIC1a pH EC50 activation

EC50 = pH 6.68 ± 0.01

C. Low pH

SyncroPatch 384i

Patchliner

384 recording channels

4 to 8 recording channels

Figure 2: Activation of ASIC1a on the SyncroPatch 384i. A: Screenshot
depicts raw data traces of ASIC1a-expressing CHO cells as recorded on
one NPC-384 patch clamp chip (single hole per well). Solution at pH 6.5
was applied to the cells (exposure time 1 s) and then washed away bycontrol solution (pH 7.2). B: pH response curve for an average of 378 wells
revealed a pH0.5 = 6.44 in excellent agreement with literature values2,3.

Optimization of ligand addition

CPatchliner

Ligand application on the Patchliner – Stacked solution

Baseline

Conc 1

pH 6.5 + DMSOConc 2pH 7.3 + DMSOConc 3pH 7.3 +test sample

A S IC 1 a - C H O S t a c k e d - t ip E C 5 0Single hole

4 hole

QC
rate

81%

94%

Rseal

1.5 GΩ

0.3 GΩ

pH 6.5 +test sample

Z’0.730.76

DEN o rm a lis e d C u rre n t

1007550EC50 = pH 6.6 ± 0.01
25-1 00500

1000

T im e (m s )1500

6 .56 .0

pHFigure 1 Activation of ASIC1a on the Patchliner. A: Schematic of addition of solution on thePatchliner using the stacked tip approach. In these experiments first wash solution (pH 7.2) andthen solution at low pH (6.0 – 7.0) were aspirated into the pipette and then rapidly applied to thecell so that the ASIC1a channels were first activated by low pH and this was quickly washed away
by control solution. B: Pipette and compound storage area of the Patchliner. C: Raw current traceof ASIC1a current during pH 6.5 application showing rapid activation, desensitization and wash-off.
D: pH response curve reveals a pH0.5 6.6 in excellent agreement with the literature2,3.

in excellent agreement with the literature2,3.

• ASIC1a could be repetitively activated with similar peak amplitudes on the Patchliner andSyncroPatch 384i enabling pharmacological studies.

• ASIC1a was blocked by benzamil and amiloride. Concentration response curves weresuccess rates for completed experiments (94% vs. 81%) and higher Z’ values (0.76 vs 0.73).

75• Conductance tracking can also be used to study ASIC1a channels on the Patchliner.

IC50 = 3.65 μM ± 1.185025References-87 .0• ASIC1a was activated with pH0.5 6.4 on the Patchliner and pH0.5 6.6 on the SyncroPatch 384i,• Either single hole or multi-hole chips can be used. The use of multi-hole chips results in higher

00or high (SyncroPatch 384i) throughput using the stacked tip approach.

constructed with IC50 values in good agreement with literature values4.

100

P e rc e n ta g e in h ib itio n0

C u rre n t (n A )• ASIC1a was reliably measured on automated patch clamp devices with medium (Patchliner)A S IC 1 a - C H O S t a c k e d - t ip IC 5 0

D-5

4 holeConc 4B
pH 6.5

Summary

SyncroPatch 384i
Single hole

BCFigure 4: Conductance tracking of ASIC1a activation on the Patchliner. A: Principle of the technique. B: Under control conditions (Vm = 0 mV)
incremental current injections assay CHO cell input resistance. C: During stacked tip application of acidic pH to activate ASIC1a receptors the Vm
responses are decreased. D: Plotting conductance changes against pH reveals an EC50 of 6.68, in good agreement with voltage clamp.

Pharmacology of ASIC1a on Patchliner and SyncroPatch 384i
AA

Repetitive activation of ASIC1a. C: Currents could be reproduciblyactivated at least 6 times with similar peak amplitudes whenrepetitively activated with low pH solution (pH 5.5).

-6-4

L o g [B e n z a m il] ( M )Figure 3: Block of ASIC1a by Benzamil onthe Patchliner. A: Pipetting scheme forapplication of inhibitor. B: Average
concentration response curve of Benzamilblock of ASIC1a mediated responses. IC50
was 3.65 µM in excellent agreement withthe literature4.

Block of ASIC1a by Benzamil or amiloride on the SyncroPatch 384i. C: Quality control data (QC rate,Rseal, Z’) were compared between single hole and multi-hole (4 holes per well) NPC-348 chips.
D: Average concentration response curve for Benzamil block of ASIC1a mediated responses on theSyncroPatch 384i. The IC50 was 1.36 µM in excellent agreement with the literature4 and with the value
obtained using the Patchliner (shown in B). E: Average concentration response curve for amiloride
block of ASIC1a mediated responses on the SyncroPatch 384i. The IC50 was 1.36 µM in excellent
agreement with the literature4.

1Lingueglia, E. & Lazdunski, M. 2013. WIREs Membr. Transp. Signal. 2:155–171
2 Wemmie, J.A., et al., 2013. Nat. Rev. Neurosci. 14(7): 461–471
3 Xu, H., et al., 2006. Nat. Neurscci. 9(5): 628-635

4 Leng, TD., et al., 2016. CNS Neurosci Ther. 22(6):468- 476.