Application Note

Channel:Cells:
Tools:P2X3CHO
SyncroPatch 384PE

Activation and inhibition of P2X3 channels
recorded on the SyncroPatch 384PE
The

electrophysiology

team

atNanion

Technologies

GmbH,Munich.

Cells engineered and kindly provided by Axxam S.p.A., Milan.

SummaryResults

P2X receptors are ligand-gated ion channels that open in

P2X3 expressed in CHO cells was activated by adding

response to extracellular ATP. They are permeable to small

ATP or ab-MeATP using the Ligand Puff function in Patch-monovalent cations, some having significant divalent orControl 384. A single concentration of ATP or ab-MeATP

anion permeability. P2X receptors are found on many cell

was applied to each well, this was repeated 3 times andtypes including smooth muscle cells, sensory neurones,

finally a maximum concentration (1 mM) ATP or ab-MeATP

epithelia, bone and leukocytes (for reviews see Refs 1 - 3).

was added to each well. The currents were normalized toA role for P2X receptors has been suggested in transmission

the maximum current in each well and the concentrationof thermal stimuli4, chemosensory signalling5, taste6,7 andresponse curve calculated across the whole plate. The

pain8,9. To date, 7 P2X receptor genes have been cloned

data was fitted using a Hill equation revealing an EC50 =and studied in heterologous expression systems (reviewed

2.04 µM (61-63 wells per concentration; 372 wells in to-in Refs 1 - 3). Functional receptors are trimeric10, whichtal), for ATP in excellent agreement with the literature2,3,12

can be homomeric or heteromeric. The P2X2 and P2X3

(Figure 1).

receptors can function either as homomers or as P2X2/3heteromers. When expressed together, a mixture of P2X2and P2X3 homomers as well as P2X2/3 heteromers are

1.0

biophysical and pharmacological properties. Both P2X3

0.8

implicated in nociception and pain signalling and may

0.6

likely to exist, which may be distinguished through theirbe important therapeutic targets for analgesic drugs8.
Additionally, the P2X3 and P2X2/3 receptor antagonist MK-I/Imaxhomomers and P2X2/3 heteromeric receptors have beenATPEC50 = 2.04 µMn = 61-63 per conc
(372 wells in total)

ATP
1 µM
3 µM
10 µM
30 µM

0.4

7264 (gefapixant), has recently progressed to Phase III
trials for refractory or unexplained chronic cough11.

0.2

Here we present data collected on the Syncro-

0.0

Patchof384PE

P2X3

showing

currents

activation

expressedinandCHO

500 msinhibition
cells

2 nAwith

rapid and brief application of ligand (‘Ligand Puff’). ATPor ab-methylene ATP (ab-MeATP) activated P2X3 receptors with an EC50 value similar to values found in the literature2,3,12. P2X3 receptors could be repetitively activatedby ATP and blocked by A-317491 with an IC50 value ingood agreement with the literature13.

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10-7

10-6

10-5
[ATP (M)]

10-4

Figure 1: Concentration response curve for ATP activation of P2X3 expressedin CHO cells. Single concentrations of ATP were added to each wellfollowed by a saturating concentration of ATP (1 mM) to calculate themaximum peak amplitude. The data was normalized to the maximum foreach well and the average concentration response curve constructedacross the whole plate revealing an EC50 of 2.04 µM. Average traces arealso shown in the inset.

Application NoteCurrents

activated

For pharmacology experiments, a stable and reproducible

by ab-MeATP (Figure 2) with an EC50 = 1.89 µM, inmediated

byP2X3 werealso

peak amplitude is mandatory. We repetitively activatedexcellent agreement with the literature

2,3,12. Given the fast

desensitizing kinetics of the currents, coupled with theP2X3 six times with 10 µM ab-MeATP (Figure 3). Peak amplitude was highly reproducible, varying with each applica-activation by ab-MeATP, we can confirm that the currents

tion by no more than 10%, demonstrating the suitability ofare mediated by P2X3 receptors since P2X2 homomeric

the Syncro-Patch 384PE for pharmacology experiments.

receptors are insensitive to ab-MeATP1-3.

1.0

0.6

mediated responses. ab-MeATP was applied 3 times tocheck for stability followed by pre-incubation with A-317491
300 nM
1 µM
3 µM
10 µM
30 µM
100 µM

0.8I/Imax

We used the P2X3 selective inhibitor A-317491 to block P2X3-αß-MeATP
EC50 = 1.89 µM (n = 56-64 per concentration)(373 wells)αß-MeATPpoint concentration response curves, where a single concentration of A-317491 was applied to each well and theconcentration response curve constructed over the wholeplate, were performed. The IC50 for A-317491 was 92 nM in
excellent agreement with the literature13.

0.4
0.2

1.0

4 nA
500 ms

0.0
10-7

2

3 4 5623 4 5610-610-5[αß-MeATP (M)]

23 4 56

10-4

Figure 2: Activation of P2X3 expressed in CHO cells on the SyncroPatch
384PE by ab-MeATP. Single concentrations of ab-MeATP were added toeach well followed by a saturating concentration of ab-MeATP (100 µM) tocalculate the maximum peak amplitude. The data was normalized to themaximum for each well and the average concentration response curve
constructed across the whole plate revealing an EC50 of 1.89 µM. Average
traces are also shown in the inset.

Aand then co-application of A-317491 with ab-MeATP. Single0.8Normalized block1.20.6A-317491
IC50 = 92 nM (n = 50-67 per concentration)(336 wells)
Control
1 nM A-317491
10 nM
100 nM
1 µM
10 µM
100 µM
A-317491
10 µM αß-MeATP

0.4

αβ-MeATP0.2
2 nA

500 ms

0.0
10-10

B

0

10-6
10-7
10-8
[A-317491 (M)]

10-5

10-4

2 nA-2Imin (nA)

10-94s-4-6-8-10500

1000

1500 2000
Time (s)

2500

3000

Figure 3: Stability of P2X3 recorded on the SyncroPatch 384PE. A. Raw current
traces from an example cell showing current activation by application of10 µM ab-MeATP repeated 6 times in the same well. B. Online analysis plot
showing peak amplitude versus time for the same well shown in A. The peak
amplitudes were very similar with each application of ab-MeATP, differing
by no more that 10% from the previous application.

Download more Application Notes from www.nanion.deFigure 4: Inhibition of P2X3 by A-317491. A-317491 was pre-incubatedfor 3 - 5 mins followed by co-application with 10 µM ab-MeATP. A single
concentration of A-317491 was applied to each well and the averageconcentration response curve for all wells was constructed giving an IC50 of92 nM. 336 wells were used to construct the concentration response curve
giving a success rate of 88% for completed experiments.

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot depicts online analysis (peakamplitude) of P2X3-expressing CHO cells as recorded on one NPC-384 patch clamp chip. At the start of the experiment, 3 control applications of ab-MeATP(10 µM) were made 10 mins apart to check for current stability. This was followed by pre-incubation of A-317491 for 3-5 mins and then co-application ofab-MeATP and A-317491. Single concentrations of A-317491 were added to each well and the concentration response curve was calculated across thewhole plate. Multi-hole chips were used where 4 holes were present per well. The data of the 384 well plate representation in the upper left part are colorcoded for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb > 30MΩ), blue (Rmemb 1.4-30 MΩ), light blue (Rmemb<1.4 MΩ) or grey (well disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show
online analysis data (peak amplitude) of P2X3 channels following activation first by ab-MeATP followed by co-application with A-317491.

Exp end and chipunload

5 min

20 - 45 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 3 control applications (-MeATPor ATP) made 5-10 mins apart, 1 compound co-application
with pre-incubation (3 - 5 mins) or full activation

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) for asingle point concentration response curve with control applications on P2X3-mediated currents took approximately 27 - 45 min.

Download more Application Notes from www.nanion.deApplication Note
Figure 5 shows a screenshot of the SyncroPatch 384
software during an experiment. A color-coded over-In conclusion, P2X3 expressed in CHO cells can bereliably and reproducibly recorded on the SyncroPatchview (based on seal resistance in this case) of all

384PE. The timeline of each experiment was approxi-384 wells gives the user a good impression of themately 30 - 45 minutes (start – end) and included 3 control

success rate of the experiment. The user can easily

applications of ab-MeATP followed by co-application

toggle between raw traces and online analysis. In thewith an inhibitor. The inhibitor (A-317491) was pre-example shown, online analysis values are chosen whereincubated for 3 - 5 mins and then co-applied with ab-peak amplitude in control conditions (10 µM ab-MeATP)

MeATP. The P2X3-mediated response was inhibited byand when co-applied with A-317491 are shown. In theseexperiments, 3 control applications of ab-MeATP areperformed followed by co-application of a single
concentration of A-317491 in each well and the concentration response curve was calculated across the wholeA-317491in a concentration-dependent manner with an
IC50 in excellent agreement with the literature13. The EC50

values for ATP and ab-MeATP and the kinetics of desensitization were consistent with P2X3 homomers1-3,12.

plate. The cells were stable for over 45 minutes, control

The SyncroPatch 384PE is a high throughput and highly

applications were made 10 minutes apart to ensure fullreliable

recovery after each application and compound wasrecording

pre-incubated for up to 5 minutes ensuring that full effectfriendlyof the compound was reached. An individual well can beadditions

highlighted to monitor progression of the experiment andeach well and easy analysis result in reliable highis shown enlarged at the bottom of the screen.

quality data at an increased throughput with anautomated
P2X3

software,orpatchclamp

receptor-mediatedexcellent

multiple

devicecurrents.

User-rates,single

success

additionsforofcompoundto

economical cost per data point.

References
1. Ralevic & Burnstock., 1998. Pharmacol. Rev. 50: 413-492
2. North & Suprenant., 2000. Annu. Rev. Pharmacol. Toxicol. 40: 563-80
3. Khakh et al., 2001. Pharmacol. Rev. 53: 107-118
4. Shimizu et al., 2005. Pain. 116: 96-108

Cell culture
Cells were cultured and harvested according toNanion’s standard cell culture protocol.
Electrophysiology

5. Prasad et al., 2001. J. Physiol. 537.3: 667-677

Whole cell patch clamp recordings were conducted

6. Finger et al., 2005. Science. 310: 1495-1499

according to Nanion’s standard procedure for the

7. Eddy et al., 2009. Chem. Senses. 34: 789-797

SyncroPatch 384PE using multi-hole (4 holes per well)

8. Chizh & Illes., 2000. Pharmacol. Rev. 53(4): 553-568

chips. Cells were held at -80 mV for the duration of the

9. North. 2003. J. Physiol. 554.2: 3016-3028

experiment. For ATP concentration response curves, 3U/

10. Nicke et al., 1998. EMBO J. 17(11): 3016-3028
11. Richards et al., 2019. Br. J. Pharmacol. doi: 10.1111/bph.14677
12. Pratt et al., 2005. J. Neurosci. 25(32): 7359-7365

ml hexokinase was used with higher concentrations ofATP. For pharmacology experiments, cells were preincubated in A-317491 for 3 - 5 min and then compound

13. Jarvis, et al, 2002 PNAS 99(26): 17179-17184

was co-applied with 10 µM ab-MeATP. P2X3 was activatedMethods

using the ‘Ligand Puff’ command in PatchControl 384.
Agonist volume: 3 µl, wash volume: 7 µl, application
speed 40 µl/s. Cells were then washed twice with external

Cells
CHO cells expressing P2X3 were engineered and kindly

solution before re-application of agonist.

provided by Axxam S.p.A., Milan; (https://axxam.com/).

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