Activation and inhibition of assay-ready TRPA1 and TRPV cells: an automated patchclamp study
Andrea Brüggemann1, Tom A. Goetze1, András Horváth1, Ilka Rinke-Weiß1, Nadine Becker1, Alison Obergrussberger1, Lukas Focke2, Oliver Wehmeier2, Ralf Schwandner3,Niels Fertig1
1Nanion Technologies, Munich, Germany; 2acCELLerate, Hamburg, Germany; 3Assay.Works, Regensburg, Germany• Records both time course and voltage
Introduction
dependence of compound application•Calculation of crude Vhalf is possible for eachsweep● www.nanion.de ● info@nanion.de ●Activation and inhibition of TRPA1 using high throughput APC
A
100 µM

Transient Receptor Potential (TRP) channels
widelyis also
distributed• Tau ofare
activation
available throughout
for different
voltages throughout
recordingthe mammalian central and peripheral nervous
systems. theThey
can be directlyactivated by ligands, heat or cold and mechano-stimulation, and areimportant targets in drug discovery for the treatment of pain, respiratory
diseases, cancer, immune disorders and others. Here, we studied theresponses of TRPA1, TRPV1, TRPV3, TRPV4 and TRPM8 assay-ready and cultured
cells activated using a variety of stimuli on automated patch clamp (APC)
systems.

CCarvacrol
200 µM

300 µM

400 µM

500 µM

Ligand activation of TRPV1 and TRPV3
TRPV1

TRPV3

600 µM

HCN2 Pharmacology in High Throughput

DB

Port-a-Patch

One recording channel

Patchliner

4 to 8 recording channels

Ligand activation

SyncroPatch 384i

384 recording channels

TRPV1
TRPV3

TRPM8 – activation by cold solution

8/17 (capsaicin)
12/12 (2-APB)

Figure 4: Activation and inhibition of TRPV1 and TRPV3 on the Patchliner. TRPV1 expressed in CHO cells was activated by capsaicin and blocked bycapsazepine (left). TRPV3 was activated by 2-APB and blocked by ruthenium red on the Patchliner (right). TRPV1-mediated currents could be
activated by capsaicin in 8/17 cells tested. TRPV3 was activated by 2-APB in all 12/12 cells tested.

Figure 2: Activation of TRPA1 expressed in CHO cell on the SyncroPatch
384i. Top panel shows graphical user interface of the screening and data
analysis software used on the SyncroPatch 384i. A 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 wasadded to each well and the concentration response curve calculated
across the plate. B Average CRC for carvacrol for n = 355 wells (left) andaverage traces. TRPA1 was robustly activated by carvacrol as expected3,4.
At higher concentrations (above 400 µM) desensitization of the channel
was observed (tachyphylaxis) as previously reported3 which resulted insmaller amplitudes upon repeated application.

Heat activation of TRPV1 and TRPV3

Inhibition of TRPA1 by A967079 and AMG0902. C TRPA1-mediatedresponses were blocked by A967079 in a concentration-dependent
manner with an IC50 value (12.4 nM) in good agreement with theliterature value of 50 nM5. D TRPA1-mediated responses were blockedby AMG0902 in a concentration-dependent manner with an IC50
value of 48 nM (n = 349) in good agreement with the literature valueof 68 nM6.

TRPV1

TRPV3

Heat activation (45°C)

All experiments were performed in perforated patch mode using
multi-hole (4 holes per well) NPC-384 chips.

TRPV1TRPV3

46/58
9/21

Activation and inhibition of TRPV4ABFigure 5: TRPV1 and TRPV3 were activated by heated solution. TRPV1 expressed in CHO cells was activated by heated solution (45°C) in 46/58 cells
tested (left). TRPV3 expressed in CHO cells was activated by heated solution in 9/21 cells tested (right). Upon repeated activation with heatedsolution, currents became larger. This sensitization of TRPV3 to repeated ligand or temperature exposure has been reported previously10,11 and is aphenomenon we have observed in previous experiments12.

Summary• 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.
• TRPA1 was robustly activated by carvacrol on a high throughput APC device (SyncroPatch 384i) with an EC50 in•••
GSK 1016790A activation Heat activation (45°C)
Figure 1: TRPM8 activation by cold solution and block by capsazepine recorded on the Port-aPatch with temperature controlled external perfusion system. A Activation of TRPM8 expressed inCHO cells using a voltage ramp (-100 mV to +100 mV over 200 ms) by cold solution (10°C; darkblue) and subsequent block by warm solution (35°C; light blue). B Corresponding timecourse of theexperiment. C Activation of TRPM8 by cold solution (10°C; dark blue) and subsequent block byeither 50 µM capsazepine (red) or warm solution (35°C; light blue). TRPM8 was reactivated by coldsolution following block by capsazepine (grey). 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 the literature1,2.

8/49

34/56

Figure 3: TRPV4 activation and inhibition on the Patchliner. A TRPV4 expressed in CHO cells were activated by the specific and potent TRPV4 agonist
GSK1016790A7,8 and this effect could be blocked by GSK2193874, a specific antagonist of TRPV49. 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.

•

good agreement with the literature. TRPA1-mediated responses were blocked by A967079 and AMG0902 asexpected.
TRPV4 was activated by GSK1016790A and blocked by GSK2193874 on the Patchliner. TRPV4 was also moderatelyactivated 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 was blocked byruthenium red.
TRPV3 sensitized upon repeated activation by heated solution.

References
1 Behrendt, H.J., et al., 2004. Br. J. Pharmacol. 141, 737– 745
2 Malkia, A., et al., 2009. Mol. Pain. 6: 62

3 Xu, H., et al., 2006. Nat. Neurscci. 9(5): 628-635
4 Saito, S., et al. 2012. JBC. 287(36): 30743-30754
5 Chen, J., et al., 2011. PAIN 152: 1165–1172

6 Lehto, S.G., et al., 2016. Mol. Pain. 12: doi: 10.1177/1744806916677761

7 Thorneloe, K.S. et al, 2008. J. Pharmacol. Exp. Ther. 326 (2): 432–442
8 Darby, W.G., et al. 2016. Int. J. Biochem. Cell. Biol. 78: 217.228
9 Cheung, M., et al. 2017. ACS Med. Chem. Lett. 8: 549−554
10 Peier et al., 2002. Science. 296: 2046-2049
11 Xu et al., 2002. Nature. 418: 181-186

12 ‘Heat activation of TRPV3 on Nanion´s Patchliner’

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