Application Note

Channel:Cells:
Tools:

P2X2/3
CHO
SyncroPatch 384PE

Pharmacology of P2X2/3 channels recordedon the SyncroPatch 384PE
The

electrophysiology

teamatNanion

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

P2X2/3 expressed in CHO cells was activated by adding

response to extracellular ATP. They are permeable to small

ATP using the Ligand Puff function in PatchControl 384. A

monovalent cations, some having significant divalent or

single concentration of ATP was applied to each well, thisanion permeability. P2X receptors are found on many cell

was repeated 3 times and finally a maximum concentra-types including smooth muscle cells, sensory neurones,tion (1 mM) ATP was added to each well. The currents

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

were normalized to the maximum current in each well

A role for P2X receptors has been suggested in transmission

and the concentration response curve calculated acrossof thermal stimuli4, chemosensory signalling5, taste6,7

the whole plate. The data was fitted using a Hill equa-and pain8. To date, 7 P2X receptor genes have beention revealing an EC50 = 1.1 µM (191 wells), in excellent

cloned and studied in heterologous expression systems

agreement with the literature2,3,10,11 (Figure 1). Only half9(reviewed in Refs 1 - 3). Functional receptors are trimeric ,the plate was used for this experiment giving a successwhich can be homomeric or heteromeric. The P2X2 andrate for completed experiments of 99% (191 out of a pos-P2X3 receptors can function either as homomers or as

P2X2/3 heteromers. When expressed together, a mixture

sible 192 wells).
AATP

Bof P2X2 and P2X3 homomers as well as P2X2/3 heteromers
are likely to exist, which may be distinguished throughI/Imaxtheir biophysical and pharmacological properties. P2X2/3

receptors have been implicated in nociception and pain
signalling and may be important therapeutic targets foranalgesic drugs8.
Here we present data collected on the SyncroPatchof384PE

P2X2/3showing

currents

activation

expressedinandCHO

inhibition
cells

with

rapid and brief application of ligand (‘Ligand Puff’).
ATP activated P2X2/3 receptors in a concentrationdependent manner with an EC50 value similar to thosereported in the literature for a mixture of homomericandheteromeric

P2X2/3

receptors2,3,10,11.

P2X2/3

receptors could be repetitively activated by ATPandblockedby

suraminwith

anIC50ingood

agreement with the literature (reviewed in refs 2 & 3).
Download more Application Notes from www.nanion.de

5 nA
1s0 mM
100 nM
300 nM
1 µM
10 µM
100 µM
1 mM

ATP1.0 EC = 1.1 µM (n = 191)
50Hill = 0.52
0.8
0.6
0.4
0.2
0.0

10-9

10-7
10-5
[ATP (M)]

10-3

Figure 1: Activation of P2X2/3 expressed in CHO cells on the SyncroPatch
384PE by ATP. A. A single concentration of ATP was applied to each well andthe average traces of all 191 cells are shown. The concentration response
curve was calculated across the plate and is shown in Panel B. The data wasnormalized to the highest response for each well. The single concentrationof ATP was applied 3 times followed by a maximum concentration of ATP(1 mM) for the normalization. A Hill equation was fit to the curve revealing
an EC50 of 1.1 µM (n = 191) in excellent agreement with the literature2,3,10,11.

Application Note
Currents mediated by P2X2/3 were also activated by a,b-For pharmacology experiments, a stable and reproducible

methylene ATP (abmeATP; Figure 2). Since P2X2 homomericpeak amplitude is critical (Figure 3). For each of the experi-receptors are insensitive to abmeATP1-3, and the kinetics

ments, 3 control applications of ATP were made beforeof desensitization are slow rather than rapid, which would

incubation in the compound (suramin). The control

be indicative of P2X3 homomeric receptors , we canapplications of 10 µM ATP showed a reproducible peakconclude that the majority to the receptors expressed inamplitude, where peak amplitude varied with eachthese cells are P2X2/3 heteromers.
ABapplication of no more than 5%. This proves the suitability of1I/Imax

βmeATP0 mM
100 nM
300 nM
1 µM10 µM
30 µM
100 µM
500 µM

2 nA
1s

the SyncroPatch 384PE for pharmacology experiments.

β-methlyene ATP
1.0 EC50 = 8.3 µM (n = 190)
Hill = 0.75
0.8For

0.6

concentrations of suramin were applied to each wellexperiments,

thenpreThree

0.9 µM (n = 372), in excellent agreement with the literature2,3. The success rate for completed experiments was

10-3
10-7
10-510-9[β-methylene ATP (M)]

97% and the cells were stable for over 30 minutes. A single
point concentration response curve, where a single concentration of suramin was applied to each well and theconcentration response curve constructed over the wholeplate, was also performed. The IC50 for suramin was similar(29 µM, n = 342) using this approach compared with thecumulative concentration response approach.
1.010 µM ATP

0.8Control
1.5 µM
15 µM
150 µM

10 µM
ATPNormalized Block2s
10 µM
ATP

Ipeak (nA)0-2-4-6-8-10-12-14

wasATP.

well to be constructed (Figure 4). This gave an IC50 = 17.7 ±0.0

10 µM
ATPwith0.22 nABsuramin

co-applied

allowing full concentration response curves for each

10 µM ATP

10 µM ATPand0.4

Figure 2: Activation of P2X2/3 expressed in CHO cells on the SyncroPatch
384PE by ab-methylene ATP. A. A single concentration of ATP wasapplied to each cell and the average traces of all 190 cells are shown.
The concentration response curve was calculated across the plate and isshown in Panel B. The data was normalized to the highest response for eachwell. The single concentration of abmeATP was applied 3 times followed
by a maximum concentration of abmeATP (500 µM) for the normalization.
A Hill equation was fit to the curve revealing an EC50 of 8.3 µM (n = 190) ingood agreement with the literature1-3.

Apharmacology

incubated

0.62 nA

0.4

1s

0.2
0

200

400

600

800
Time (s)

1000

1200

1400

Suramin
IC50 = 17.7 ± 0.9 µM (n = 372)

0.0
Figure 3: Stability of P2X2/3 recorded on the SyncroPatch 384PE. A. Raw
current traces from an example cell showing current activation byapplication of 10 µM ATP repeated 3 times in the same cell. B. Onlineanalysis plot showing peak amplitude versus time for the same cell shown
in A. The peak amplitudes were very similar with each application of ATP,differing by no more that 5%.

10-8

10-7

10-6

10-5

10-4

10-3

10-2

[Suramin (M)]
Figure 4: Inhibition of P2X2/3 by suramin. Suramin was pre-incubated at eachconcentration for 3 - 5 mins followed by co-application with 10 µM ATP. All
3 concentrations of suramin were applied to each well and a cumulative
concentration response curve was constructed for n = 372 wells out of apossible 384. Success rate was 97% for completed experiments.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot depicts raw data traces of P2X2/3expressing CHO cells as recorded on one NPC-384 patch clamp chip. A cumulative concentration response curve for suramin was performed on eachwell. Three control applications of 10 µM ATP were made followed by 3 increasing concentrations of suramin (1.5, 15 and 150 µM) on each well. Suramin
was pre-incubated 3 - 5 mins followed by co-application of ATP. Multi-hole chips were used where 4 holes were present per well. The data of the 384 wellplate representation in the upper left part are color-coded for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb >10MΩ) or blue (Rmemb <10 MΩ). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show
raw data traces of P2X2/3 channels following activation first by ATP followed by co-application with suramin.

Exp end and chipunload
20 - 45 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 3 control applications (ATP), 1 - 3compound co-applications with pre-incubation (3 - 5 mins)

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 a
1 - 3 point concentration response curve on P2X2/3-mediated currents took approximately 27 - 45 min.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application Note
Figure 5 shows a screenshot of the SyncroPatch 384

mately 30 - 45 minutes (start – end) and included 3 control

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

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

with384 wells gives the user a good impression of theinhibitor (suramin) was pre-incubated for 3 - 5 mins

success rate of the experiment. The user can easily

and then co-applied with ATP. The P2X2/3-mediatedtoggle between raw traces and online analysis. In
the example shown, raw traces are chosen and thegraphs show control responses to 10 µM ATP followed
by inhibition by co-application of suramin. The experiment shows that a full concentration response curve
can be performed on each well, thus reducing the cost

1-of
3ATP

followed

concentrations

ofby

co-application

aninhibitor.

Theresponse was inhibited by suramin in a concentrationdependent manner as expected with an IC50 within therange round in the literature2,3. The EC50 values for ATP
and abmeATP and the kinetics of desensitization wereconsistent with P2X2/3 heteromers1-3,10,11.

per data point. The cells were stable for over 30 min-The SyncroPatch 384PE is a high throughput and highlyutes and compound could be pre-incubated for up toreliable

5 minutes per concentration ensuring that full effect ofrecording

the compound was reached. An individual well can befriendly

highlighted to monitor progression of the experiment andadditions

is shown enlarged at the bottom of the screen.

each well and easy analysis result in reliable highautomated
P2X2/3software,
or

patch

clamp

receptor-mediatedexcellent

multiple

success

additionsofdeviceforcurrents.

User-rates,

singlecompoundto

quality data at an increased throughput with an
In conclusion, P2X2/3 expressed in CHO cells can be

economical cost per data point.

reliably and reproducibly recorded on the SyncroPatch

384PE. The timeline of each experiment was approxi-References

Cell culture
Cells were cultured and harvested according to

1. Ralevic & Burnstock., 1998. Pharmacol. Rev. 50: 413-492

Nanion’s standard cell culture protocol.

2. North & Suprenant., 2000. Annu. Rev. Pharmacol. Toxicol. 40: 563-80

Electrophysiology

3. Khakh et al., 2001. Pharmacol. Rev. 53: 107-118

Whole cell patch clamp recordings were conducted

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

according to Nanion’s standard procedure for the

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

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

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

chips. Cells were held at -80 mV for the duration of the7. Eddy et al., 2009. Chem. Senses. 34: 789-797

experiment. For ATP concentration response curves, 3U/

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

ml hexokinase was used with higher concentrations of

9. Kawate et al., 2011. J. Gen. Physiol. 137(6) 579–590

ATP. For pharmacology experiments, cells were pre-10. Ueno et al., 1998. Br.J. Pharmacol. 124: 1484-1490

incubated in suramin for 3 - 5 min and then compound

11. Liu et al., 2001. JPET. 296: 1043-1050

was co-applied with 10 µM ATP. P2X2/3 was activatedusing the ‘Ligand Puff’ command in PatchControl 384.

Methods

Agonist volume: 3µM, wash volume: 7 µM, application
speed 40 µl/s. Cells were then washed twice with external

Cells

solution before re-application of agonist.

CHO cells expressing P2X2/3 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