Kinetic and pharmacological properties of P2X3 and P2X2/3
receptors
James Costantin1, Tim Strassmaier1, Giustina Rotordam2, Tom Goetze2, Nadine Becker2, Alison Obergrussberger2, Andrea
Brüggemann2, Michael George2, Niels Fertig2
1Nanion Technologies Inc, Livingston, NJ, USA, 2Nanion Technologies, Munich, Germany.

Introduction

Pharmacology of P2X2/3 channels: High throughput activation, stability and inhibition

P2X receptors are ligand-gated ion channels activated byextracellular ATP. They are permeable to small monovalentcations, some having significant divalent or anion permeability.
The P2X2 and P2X3 subunits are predominantly expressed in primarysensory neurons and have been proposed to play a role in thermal
sensation, taste and pain. They form functional hetero- or
homotrimers which are activated by ATP or αβ-methylene ATP (αβMeATP). The stoichiometry of P2X2/3 heteromers appears to bedependent on the relative abundance of the two subunits. A
mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers
are likely to exist, which can be distinguished through theirbiophysical and pharmacological properties. The receptors open
in response to an increase in extracellular ATP which occurs underpathological conditions such as tissue damage. The resulting
depolarization leads to propagation of the pain signal. Due to itsrole in nociception and pain signaling, these receptors are
considered to be important targets for pain management.
Here we present data collected on automated patch clamp(APC) systems showing activation and inhibition of P2X2/3 and P2X3
receptors expressed in CHO cells with rapid and brief applicationof ligand and pre-incubation of inhibitors.

activationActivation of P2X2/3 expressed in CHO cells on theSyncroPatch 384PE by αβ-methylene ATP (αβ-MeATP). Asingle concentration of αβ-MeATP was applied to eachwell and the average traces of all 190 wells are shown.
The concentration response curve was calculated acrossthe plate and is shown. The data was normalized to thehighest response for each well. The single concentrationof αβ-MeATP was applied 3 times followed by amaximum concentration of αβ-MeATP (500 µM) for thenormalization. A Hill equation was fit to the curverevealing an EC50 of 8.3 µM (n = 190) in good agreementwith 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 shown here,
activation by αβ-MeATP is consistent with activation ofP2X2/3 heteromers and not P2X3 homomers (see Ref. 4and data below).

inhibition

reproducibility

Stability of P2X2/3 recorded on the SyncroPatch 384PE:
Raw traces showing current activation by 10 μM ATP
repeated 3 times in the same cell (upper panel). Online
analysis plot (bottom panel) shows stable peak
amplitudes versus time, differing by no more than 5%.

Workflow of the experiment: Three control applications of
10 μM ATP were made followed by 3 increasing
concentrations of suramin (1.5, 15 and 150 μM) on eachwell. Suramin was pre-incubated for 3 - 5 mins followedby co-application of ATP. The experiment tookapproximately 25 – 50 mins depending on number ofapplications.

Pharmacology of P2X3 channels: Activation, stability and inhibition
Effect of applying seal compensation and simulated IK1 to hiPSC-cardiomyocytes on AP shape and pharmacology

Inhibition of P2X2/3 by suramin: All 3 concentrations ofsuramin were applied to each well and a cumulative
concentration response curve was constructed for (n =372 of 384). Success rate was 97% for completedexperiments. The IC50 was in good agreement with values
found in the literature2-4.
Graphical user interface of the screening and data analysis software used on theSyncroPatch 384PE: Screenshot depicts raw data traces (above) of P2X2/3-expressing
CHO cells as recorded on one NPC-384 patch clamp chip (4 holes per well). A
cumulative concentration response curve for suramin was performed on each well.

SyncroPatch 384PE

activation

Patchliner

inhibition

reproducibility

Stability of P2X3 recorded on the
SyncroPatch 384PE. Raw tracesfrom CHO cells expressing P2X3
showing current activation by 10μM αβ-MeATP repeated 5 times
in the same cell (upper panel).
Online analysis plot (middlepanel)showsstablepeak
amplitudes (average of 93 wells)
versus time, differing by no morethat 8% from the previous peak.
Seal resistance is stable during
the course of the experiment(lowerpanel),
shown
are
average Rseal values of 93 wells
versus time.
Multi-hole chips
were used for the experiments (4holes per well).

Conclusions
Ion channel currents mediated by P2X2/3 heteromers expressed in

CHO cells were activated by ATP and αβ-MeATP on the SyncroPatch
384PE at high throughput.
P2X2/3 heteromeric receptors were inhibited by suramin in aconcentration-dependent manner with expected IC50.
P2X3 homomers were successfully recorded on the Patchliner andSyncroPatch 384PE, displaying faster activation and inactivation
kinetics compared to the P2X2/3 heteromers.
Currents mediated by P2X3 were highly reproducible, activated byATP or αβ-MeATP.
P2X3-mediated currents were blocked by the specific inhibitor A317491.
Patchliner and SyncroPatch 384i can be used to measure different
P2X-mediated currents and test compounds for specificity.
Inhibition of P2X3 by A-317491. P2X3 was inhibited by the P2X3-selective compound,A-317491, on both the Patchliner and SyncroPatch 384PE when activated by 10 µM
αβ-MeATP with similar IC50 values and in good agreement with the literature5.

Left: Activation of P2X3 expressed in CHO cells by ATP and αβ-MeATP. Top:
Activation of P2X3 by ATP on the SyncroPatch 384PE with an EC50 of 2.04 µM, ingood agreement with the literature2-5. Lower panel: Activation of P2X3 by αβMeATP on the Patchliner with an EC50 of 5.6 ± 0.3 µM (n = 12). A similar EC50 wascalculated on the SyncroPatch 384PE (1.89 µM, n = 373) for αβ-MeATP (notshown). These values are also in good agreement with the literature2-4,6.

Data generated on the SyncroPatch 384PE, a predecessor model of theSyncroPatch 384i.
Cells kindly provided by Axxam S.p.A

Data generated on SyncroPatch 384PE, a predecessor model of theSyncroPatch 384i
Cells kindly provided by Axxam S.p.A

References
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5. Jarvis, et al., 2002. PNAS 99(26): 17179–17184
6. Pratt, et al., 2005. J. Neurosci. 25(32):7359–7365