Pharmacology of P2X3 and P2X2/3 receptors in cell lines and hiPSC-derived
neurons: An automated patch clamp study
Elena Sirtori1, Jean-Francois Rolland1, Riccardo Rizzetto1, Elena-Laura Budusan2, Jan Rathje3, Maria Giustina Rotordam3, Jan Rathje3, Tom A. Goetze3, Nadine Becker3, Alison Obergrussberger3,Irene Lu4, Tim Strassmaier4, Aaron Randolph4, Vincent Truong5, Patrick Walsh5, Niels Fertig3
Axxam SpA, Milan, Italy, 2University of Queensland, Brisbane, Australia; 3Nanion Technologies, Munich, Germany; 4Nanion Technologies Inc, New Jersey, USA, 5Anatomic Inc., Minneapolis, MN, USA
ali@nanion.de, Jeanfrancois.rolland.jr@axxam.com, vince@anatomic.tech

12Introduction

P2X receptors are ligand-gated ion channels activated by extracellular ATP. They
are permeable to small monovalent cations, some having significant divalent oranion permeability. The P2X2 and P2X3 subunits are predominantly expressed inprimary sensory neurons and have been proposed to play a role in thermal
sensation, taste and pain. They form functional hetero- or homotrimers which areactivated by ATP or αβ-methylene ATP (αβ-MeATP). The stoichiometry of P2X2/3
heteromers appears to be dependent on the relative abundance of the twosubunits. A mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers arelikely to exist, which can be distinguished through their biophysical andpharmacological properties. The receptors open in response to an increase in
extracellular ATP which occurs under pathological conditions such as tissue
damage. The resulting depolarization leads to propagation of the pain signal.
Due to its role in nociception and pain signaling, these receptors are considered
to be important targets for pain management.

Cross-platform comparison of pharmacology of P2X2/3 andP2X3 channels

ABDC

Instrument

αβ-MeATP EC50 (µM)

Instrument

Suramin IC50 (µM)

Instrument

αβ-MeATP EC50 (µM)

A-317491 IC50 (nM)Port-a-Patch

9.5 ± 0.5 (n = 7)

Port-a-Patch

4.7 ± 1.1 (n = 14)

Patchliner

1.7 ± 0.4 (n = 5)85.9 ± 20.7 (n = 9)

Patchliner

3.7 ± 0.9 (n = 6)

Patchliner

8.7 ± 1.3 (n = 6)

SyncroPatch 384

1.9 (n = 373)

92 (n = 336)

SyncroPatch 384

8.3 (n = 190)

SyncroPatch 384

17.7 ± 0.9 (n = 372)

Table 1: EC50 values for αβ-MeATP (P2X2/3).

Table 2: IC50 values for suramin (P2X2/3).

Table 3: EC50 values for αβ-MeATP and IC50 values for A-317491 (P2X3).

Here we present data collected on three different automated patch clamp(APC) systems at two different sites showing activation and inhibition of P2X2/3
and P2X3 receptors expressed in CHO cells with rapid and brief application ofligand and pre-incubation of inhibitors. In addition, we show P2X-mediatedcurrent responses from hiPSC-derived nociceptors (RealDRGTM).

Figure 1: A. Concentration response curves (CRCs) for activation of P2X2/3 expressed in CHO cells on the Port-a-Patch, Patchliner and SyncroPatch 384 by αβ-methylene ATP (αβ-MeATP) shown overlaid
with example traces for P2X2/3 recorded on the Patchliner shown in the inset. The EC50s for αβ-MeATP (Table 1) were in good agreement with the literature1-4. αβ-MeATP activates P2X3 containing receptors
thus activating P2X3 homomeric and P2X2/3 heteromeric receptors and not P2X2 homomers2-4. Given the slow desensitization of the recordings, activation by αβ-MeATP is consistent with P2X2/3 heteromers
and not P2X3 homomers (see Ref. 4 and data in C). B. Inhibition of P2X2/3 by suramin. P2X2/3 was activated by 30 µM αβ-MeATP (Port-a-Patch and Patchliner) or 10 µM ATP (SyncroPatch 384) and the cells
were pre-incubated in suramin for 1 – 3 min prior to co-application with ligand. The IC50 values (Table 2) were all in good agreement with the literature2-4. C. Concentration response curves for activation ofP2X3 by αβ-MeATP for the Patchliner and SyncroPatch 384 are shown overlaid. The EC50 values (Table 3) showed little difference between the 2 devices and are in good agreement with the literature2-4,6.
D. P2X3 was inhibited by A-317491 (a P2X3-selective compound) on both the Patchliner and SyncroPatch 384 when activated by 10 µM αβ-MeATP with similar IC50 values (Table 3) and in good agreementwith the literature5. Example traces from the Patchliner are shown in the inset. E. Stability of P2X2/3 recorded on the SyncroPatch 384. Raw traces showing current activation by 10 μM ATP repeated 3 times inthe same cell (upper panel). Online analysis plot (bottom panel) shows stable peak amplitudes versus time, differing by no more than 5% when ATP was applied.

34A

Reproducibility and effect oftemperature on P2X3 currents
SyncroPatch 384

BPort-a-PatchAP2X receptors in hiPSC-derivedTM
nociceptors - RealDRG

SyncroPatch 384 – application of ATPBC Patchliner – application of ATP

20 mM ATP5•DD

14 DIV – 4 min recovery time

E28 DIV – 10 min recovery time

CConclusions• Ion channel currents mediated by P2X2/3 heteromers•C•••

expressed in CHO cells were activated by αβ-MeATP on thePort-a-Patch, Patchliner and SyncroPatch 384 with similar
EC50 values and inhibited by suramin with expected IC50s.
P2X3 homomers were successfully recorded on the Patchliner
and SyncroPatch 384, displaying faster activation andinactivation kinetics compared to the P2X2/3 heteromers.
Currents mediated by P2X3 were highly reproducible,activated by αβ-MeATP on the Port-a-Patch, Patchliner andSyncroPatch 384.
P2X3-mediated currents were blocked by A-317491 on thePatchliner and SyncroPatch 384 with similar IC50s.
Increased temperature caused an increase in amplitudeand faster recovery from desensitization.
P2X-mediated responses were detected in RealDRGTM
sensory neurons.

References
Figure 2: A & B. Raw traces from CHO cells expressing P2X3 showing current activation by 10 μM αβ-MeATP repeated 6 times in the same cellon the SyncroPatch 384 (A) and activation by 1 μM αβ-MeATP repeated 6 times on the Port-a-Patch (B) showing highly stable responses. P2X3currents activated by 1 μM αβ-MeATP recorded on the Port-a-Patch were larger in amplitude when heated to physiological temperaturecompared with room temperature (C, inset). Heating to physiological temperature resulted in a faster recovery of P2X3 currents when theagonist was applied in longer intervals (C) but had negligible effects on tachyphylaxis (D).

EFigure 3: A. P2X-mediated responses from hiPSC-derived nociceptors (RealDRGTM) receptors were recorded when activated using 20 µM ATP on
the SyncroPatch 384. B. Current density of ATP-activated currents decreased during maturation (further experiments are required to confirm thisobservation). C. P2X receptors in RealDRGTM were activated using 20 µM ATP on the Patchliner. D. With a recovery time of 4 min between ATP
applications, desensitization meant that currents could not be repetitively activated upon a 2nd or 3rd application of ATP. E When a recoverytime of 10 min was used, P2X could be repetitively activated 3 times within the same cell with a current amplitude similar in amplitude to the 1stapplication (D, E Experiments on the SyncroPatch 384).

1.
2.

3.
4.
5.
6.

Ralevic & Burnstock. 1998. Pharmacol. Rev. 50: 413-492
North & Suprenant. 2000. Annu. Rev. Pharmacol. Toxicol. 40:563-80
Khakh et al., 2001. Pharmacol. Rev. 53: 107-118
Dunn et al., 2001. Prog. in Neurobiol. 65: 107–134
Jarvis, et al., 2002. PNAS 99(26): 17179–17184
Pratt, et al., 2005. J. Neurosci. 25(32):7359–7365