Application Note

Channel:Cells:
Tools:P2X3CHOPatchliner

Activation and inhibition of P2X3 channels
recorded on the Patchliner
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells engineered and kindly provided by Axxam S.p.A, Milan.

SummaryResults

P2X receptors are ligand-gated ion channels that open

P2X3 expressed in CHO cells was activated by adding ab-in response to extracellular ATP. They are permeable

MeATP using the stacked solutions addition function ofto small monovalent cations, some having significant

the Patchliner. First ab-MeATP was applied to the cell anddivalent or anion permeability. P2X receptors are foundthis is quickly washed away by buffer solution also pres-on many cell types including smooth muscle cells, sensory

ent in the pipette. Multiple applications of ab-MeATP inneurones, epithelia, bone and leukocytes (for reviews see

increasing concentrations (1-300 µM) were made to eachRefs 1 - 3). A role for P2X receptors has been suggested inwell and the concentration response curve (CRC) for antransmission of thermal stimuli4, chemosensory signalling5,

average of 12 cells is shown in Figure 1. The data was fitted

taste6,7 and pain8,9. To date, 7 P2X receptor genes haveusing a Hill equation revealing an EC50 = 3.6 ± 0.3 µM

been cloned and studied in heterologous expression
systems (reviewed in Refs 1 - 3). Functional receptors are(n = 12), for ab-MeATP in good agreement with theliterature2,3,12 (Figure 1).

trimeric10, which can be homomeric or heteromeric. Theαß-MeATP
1.0 EC50 = 3.6 ± 0.3 µM (n = 12)

P2X2 and P2X3 receptors can function either as homomers
or as P2X2/3 heteromers. When expressed together, amixture of P2X2 and P2X3 homomers as well as P2X2/30.8heteromers are likely to exist, which may be distinguishedtheirbiophysicaland

αβ-MeATPpharmacologicalproperties. Both P2X3 homomers and P2X2/3 heteromeric
receptors have been implicated in nociception and pain

signalling and may be important therapeutic targets forI/Imax

through

0.6

1 µM
10 µM
30 µM
300 µM

0.4

analgesic drugs . The P2X3 and P2X2/3 receptor antagonist
8MK-7264 (gefapixant), has recently progressed to Phase

0.2

2 nA

III trials for refractory or unexplained chronic cough11.
Here we present data collected on the Patchliner showing activation and inhibition of P2X3 currents expressedin CHO cells with rapid and brief application of ligand(using the stacked solution approach). ab-methylene ATP(ab-MeATP) activated P2X3 receptors in a concentrationdependent manner with an EC50 value similar to thosein the literature2,3,12. P2X3 receptors could be repetitively

activated by ab-MeATP and blocked by A-317491 withan IC50 value in good agreement with the literature13.
Download more Application Notes from www.nanion.de0.0

200 ms
1 µM

10 µM[αß-MeATP]

100 µM

Figure 1: Activation of P2X3 expressed in CHO cells on the Patchliner by abMeATP. Cumulative concentrations of ab-MeATP were added to each well
(1-300 µM) and the data normalized to the highest concentration. The CRC
for an average of 12 cells is shown here with the raw data traces from anexemplar cell shown in the inset. The average EC50 was 3.6 ± 0.3 µM (n = 12).

Application Note
For pharmacology experiments, a stable and reprod-1.0

vated P2X3 five times with 30 µM ab-MeATP (Figure 2). Peakamplitude was highly reproducible, varying by no morethan 12% from the first application, demonstrating thesuitability of the Patchliner for pharmacology experiments.
αβ-MeATP

Normalized Block

ucible peak amplitude is mandatory. We repetitively acti-A-317491
IC50 = 85.9 ± 20.7 nM (n = 9)0.8

A-317491
10 µM αβ-MeATP

0.60.4

1 nAControl
10 nM
100 nM
1 µM
10 µM

0.2

200 ms0.0100 pM 1 nM

1 nA5s

10 nM 100 nM 1 µM

10 µM 100 µM[A-317491]

Figure 2: Stability of P2X3 recorded on the Patchliner. Raw current tracesfrom an example cell showing current activation by application of 30 µM
ab-MeATP repeated 5 times in the same well.

Figure 3: Inhibition of P2X3-mediated currents by A-317491. A-317491 waspre-incubated for 3 - 5 mins followed by co-application with 10 µM abMeATP. Full concentration response curves were performed on each celland the average concentration response curve for 9 cells is shown. Theaverage IC50 was 85.9 ± 20.7 nM (n = 9).

We used the P2X3 selective inhibitor A-317491 to block

In conclusion, the Patchliner can be used to reliably

to check for stability followed by pre-incubation withinhibition pharmacology in good agreement with theP2X3-mediated responses. ab-MeATP was applied 3 times

record P2X3-mediated responses with activation andA-317491 and then co-application of A-317491 with ab-literature. Given the putative role of P2X3-containing

MeATP. Full CRCs were performed on each cell. The CRC

receptors in inflammatory and neuropathic pain9, thefor an average of 9 cells is shown in Figure 3. The IC50 for

Patchliner could be used to identify P2X3-specific inhibitorswith the literature13.

conditions such as chronic cough.

References

Cell culture

A317491 was 85.9 ± 20.7 nM (n = 9) in excellent agreement

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

as potential novel pain therapeutics and to treat other

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

4. Shimizu et al., 2005. Pain. 116: 96-108

Whole cell patch clamp recordings were conducted

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

according to Nanion’s standard procedure for the

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

Patchliner. Cells were held at -70 mV for the duration of

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

the experiment. For ab-MeATP concentration response

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

curves, 3U/ml hexokinase was used with all concentrations

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

of ab-MeATP. For pharmacology experiments, cells were

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

pre-incubated in A-317491 for 2 min and then compound
was co-applied with 10 µM ab-MeATP. P2X3 was activatedusing the stacked solutions approach. Agonist volume: 5

13. Jarvis et al, 2002 PNAS 99(26): 17179-17184µl, wash volume: 150 µl, application speed 114 µl/s. Cells

Methods

were then washed twice with external solution before reapplication of agonist.

Cells
CHO cells expressing P2X3 were engineered and kindly
provided by Axxam S.p.A., Milan; (https://axxam.com/).
Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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