Pharmacology of transient receptor potential cation(TRP) channels using different activation stimuli
Alison Obergrussberger1, András Horváth1, Tom A. Goetze1, Ilka Rinke-Weiß1, Nadine Becker1, Lukas Focke2, Oliver Wehmeier2,Ralf Schwandner3, Niels Fertig1,1Nanion Technologies, Munich, Germany, 2acCELLerate, Hamburg, Germany, 3Assay.Works, Regensburg, Germany

Contact: ali@nanion.de1

4Introduction

Transient Receptor Potential (TRP) channels are widely distributed

TRPV1 and TRPV3 – ligand and heatBAthroughout the mammalian central and peripheral nervous systems. They
can be directly activated by ligands, heat or cold and mechano-stimulation, and are important targets in drug discovery for the treatmentof pain, respiratory diseases, cancer, immune disorders and others. Here,
we studied the responses of TRPA1, TRPV1, TRPV3, TRPV4 and TRPM8 assayready and cultured cells activated using a variety of stimuli on automatedCpatch clamp (APC) systems.

2
A

TRPM8 – activation by cold solution

Heat activation

TRPV1

8/17 (capsaicin)

46/48

TRPV3

12/12 (2-APB)

9/21Figure 3: Activation and inhibition of TRPV1 and TRPV3 on the Patchliner. A TRPV1 expressed in CHO cells
was activated by capsaicin and blocked by capsazepine. B TRPV3 was activated by 2-APB andblocked by ruthenium red. TRPV1-mediated currents could be activated by capsaicin in 8/17 cells
tested and activated by heated solution in 46/48 cells tested (C). TRPV3 was activated by 2-APB in all
12/12 cells tested and activated by heated solution in 9/21 cells tested (D).

5B

Ligand activation

High throughput activation andinhibition of TRPA1ACarvacrol
100 µM 200 µM 300 µM 400 µM 500 µM

CD600 µMCDDBEFFigure 1: TRPM8 activation by cold solution and block by capsazepine recorded on the Port-a-Patchwith temperature controlled external perfusion system. A Activation of TRPM8 expressed in CHO cells
using a voltage ramp (-100 mV to +100 mV over 200 ms) by cold solution (10°C; dark blue) andsubsequent block by warm solution (35°C; red). B Corresponding timecourse of the experiment. CActivation of TRPM8 by cold solution (10°C; dark blue) and subsequent block by either 50 µM
capsazepine (green) or warm solution (35°C; red). TRPM8 was reactivated by cold solution following
block by capsazepine (light blue). D Corresponding timecourse of the experiment. E Concentration
response curve (CRC) for capsazepine block of TRPM8 activated by cold solution. The curve was fit
with a Hill equation and an IC50 = 12.9 µM (n = 1) was calculated, in excellent agreement with theliterature1,2. F TRPM8 was also activated by decreasing temperature on the SyncroPatch 384.

3TRPV4 – activation and inhibition

GSK 1016790A activation

Heat activation (45°C)

8/49

34/56

Figure 2: TRPV4 activation and inhibition on the Patchliner. A TRPV4 expressed in CHO cells wereactivated by the specific and potent TRPV4 agonist GSK1016790A3,4 and this effect could be blocked
by GSK2193874, a specific antagonist of TRPV45. B TRPV4 expressed in CHO cells was also moderately
activated by heated solution (45°C). External solution was heated inside the pipette of the Patchliner
and rapidly applied to the cells. In 34/56 cells tested, activation by heated solution could be observed.

Figure 4: Activation and inhibition of TRPA1 expressed in CHO cell on the SyncroPatch 384. A Graphicaluser interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot
depicts raw data traces of TRPA1-expressing CHO cells as recorded on one NPC-384 patch clamp chip
(4 holes). A single concentration of carvacrol was added to each well and the concentration response
curve calculated across multiple wells. B Average CRC for carvacrol for n = 355 wells (left) and average
traces (right). TRPA1 was robustly activated by carvacrol as expected6,7. At higher concentrations(above 400 µM) desensitization of the channel was observed (tachyphylaxis) as previously reported6
which resulted in smaller amplitudes upon repeated application. C TRPA1-mediated responses wereblocked by A967079 in a concentration-dependent manner with an IC50 value (12.4 nM) in good
agreement with the literature value of 50 nM8. D TRPA1-mediated responses were blocked byAMG0902 in a concentration-dependent manner with an IC50 value of 48 nM (n = 349) in goodagreement with the literature value of 68 nM9. All experiments were performed in perforated patch
mode using multi-hole (4 holes per well) NPC-384 chips.

6Conclusions•TRP channels can be reliably measured using automated patch clamp.

•TRPM8 was activated by cold solution and this was blocked by capsazepine on the Port-a-Patch.
TRPM8 was also activated by decreasing temperature on the SyncroPatch 384.

•TRPV4 was activated by GSK1016790A and blocked by GSK2193874 on the Patchliner. TRPV4 wasalso moderately activated by heated solution (45°C).

•TRPV1 was activated by capsaicin and heated solution (45°C) and blocked by capsazepine.

•TRPV3 was activated by 2-APB and heated solution (45°C). The 2-APB activated response wasblocked by ruthenium red.

•TRPV3 sensitized upon repeated activation by heated solution.

•

TRPA1 was robustly activated by carvacrol on a high throughput APC device (SyncroPatch 384)with an EC50 in good agreement with the literature. TRPA1-mediated responses were blocked byA967079 and AMG0902 as expected.

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