Application Note

Channel:Cells:
Tools:

P2X2/31321N1Patchliner

P2X2/3 receptors recorded on Nanion´sPatchliner
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Evotec.

SummaryResults

P2X receptors are ligand-gated ion channels that open in

Current responses of an individual cell expressing

response to extracellular ATP. They are permeable to small

P2X2/3anion permeability. P2X receptors are found on many cell
types including smooth muscle cells, sensory neurones,
epithelia, bone and leukocytes (for reviews see Refs 1 - 3).
A role for P2X receptors has been suggested in transmissionof thermal stimuli4, chemosensory signalling5, taste6,7and pain8. To date, 7 P2X receptor genes have beencloned and studied in heterologous expression systems

receptors

literature for a mixture of P2X2 homomers and P2X2/3
2,3,10,11

heteromers.

2,3,10,111.0
0.1 µM
1 µM
3 µM
10 µM
30 µM
100 µM
300 µM

receptors have been implicated in nociception and pain
signalling and may be important therapeutic targets forATP activated P2X2/3 receptors in a concentration-dependent manner with an EC50 similar to thosereported in the literature for a mixture of homomericand heteromeric P2X2/3 receptors2,3,10,11. The currents

desensitized slowly confirming that they are mediatedby P2X2 and P2X2/3 receptors rather than P2X3 receptors. P2X2/3 receptors could be repetitively activatedby ATP and blocked by suramin with an IC50 in good
agreement with the literature (reviewed in refs 2 & 3).

Download more Application Notes from www.nanion.dealso.

ATPtheir biophysical and pharmacological properties. P2X2/3liner to record P2X2/3 currents activated by ATP.

isB

are likely to exist, which may be distinguished throughnel P­ atchliner showing the p­ otential use of the Patch-desensitizationmuch faster desensitization with a slow recovery fromof P2X2 and P2X3 homomers as well as P2X2/3 heteromers

Here we present data collected on a 4- or 8-chan-slow

rather than P2X3 homomers as P2X3 receptors exhibit

Aanalgesic drugs .

Theindicative of P2X2 homomers and P2X2/3 heteromers

which can be homomeric or heteromeric. The P2X2 and8concentrations

7.8 ± 1.0 µM (n = 10), in good agreement with thedesensitization

P2X2/3 heteromers. When expressed together, a mixture

increasing

response curve revealed an EC50 for ATP activation of(reviewed in Refs 1 - 3). Functional receptors are trimeric9,
P2X3 receptors can function either as homomers or as

toof ATP are shown in Figure 1. A concentration

1 nA

200 ms0.8
I/Imaxmonovalent cations, some having significant divalent or0.6
0.4
0.20.00.011[ATP (µM)]

100

Figure 1:
A Activation of P2X2/3 receptors by increasing concentrations of ATP.
B Concentration response curve for ATP activation, EC50 = 7.8 ± 1.0 µM(n = 10).

A

B

Suramin
30 µM ATP1.0
0.8

Figure 2 shows the repetitive activation of P2X2/3 receptors.

0 µM
1 µM
10 µM
100 µM
1 mM

Currents were activated with a similar peak amplitude
when challenged 7 times with 30 µM ATP.
A

0.5 nA
200 ms
2 nA5s
Ipeak (nA)B0-4

1-(I/Imax)

Application Note

0.60.40.20.00.01

1100[Suramin (µM)]

Figure 3:
Block of P2X2/3 receptors by suramin. Suramin at increasing concentrations(1 µM - 1 mM) was pre-incubated and then co-applied with 30 µM ATP.
Full recovery from block was achieved upon washout (data not shown)
B Concentration response curve for suramin block, IC50 = 28.0 ± 5.3 µM (n= 7).

-8123
45Sweep Count67

Figure 2:
A P2X2/3 receptors could be repetitively activated by 30 µM ATP. B Timecourse of the experiment. P2X2/3 currents of approx. 8 nA were activated
7 times reproducibly in the same cell.

A full concentration response curve to suramin wasperformed (Fig. 3). Suramin blocked the ATP activatedcurrent with an IC50 of 28.0 ± 5.3 µM (n = 7) in good

agreement with literature values (reviewed in refs 2 & 3).

References

In summary, P2X2/3 receptors stably expressed in 1321N1
cells can be reliably activated by ATP with EC50 values

and desensitization properties consistent with a mixture ofP2X2 homomers and P2X2/3 heteromers. The ATP activatedcurrents could also be blocked by suramin. The data
shown here agrees well with published literature using
conventional patch clamp electrophysiology to study
P2X2/3 receptors1-11. Therefore, the Patchliner provides

a viable, higher throughput alternative to conventional
patch clamp for the discovery of active P2X2/3 leadcompounds.

Cell culture

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

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 a holding potential of

7. Eddy et al., 2009. Chem. Senses. 34: 789-797-70 mV. To achieve short exposure times, solutions were

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

stacked in the robotic pipettor. First, wash solution (155 µl)

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

was aspirated followed by aspiration of the agonist-

10. Ueno et al., 1998. Br.J. Pharmacol. 124: 1484-1490

containing solution (40 µl) and then applied to the cell at

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

a speed of 57 µl/s. Wash solutions contained theantagonist in the suramin experiments. The cells wereMethods

pre-incubated in suramin before co-application with
30 µM ATP.

Cells
1321N1 cells stably expressing P2X2/P2X3 subunits, kindly
provided by Evotec, were used.

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