Application NoteChannel:
Cells:
Tools:hTRPA1
CHO
SyncroPatch 384

Reproducible activation and pharmacologyof hTRPA1 on the SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly supplied by Charles River.

SummaryResults

The transient receptor potential ankyrin 1 (TRPA1) is a

Human TRPA1 expressed in CHO cells was activated bycalcium permeable non-selective cation channel thatcarvacrol in a concentration-dependent manner (Fig.

belongs to the transient receptor potential (TRP) super-1). Carvacrol, a major component of oregano, has beenfamily. The TRPA1 channel is expressed in the sensory

shown to activate TRPV3 and TRPA1 channels12. TRPA1

neurons of the nodose ganglia, dorsal root ganglia, andcurrents activated by carvacrol quickly desensitize in thetrigeminal ganglia1-3, and also non-neuronal cells suchcontinued presence of the agonist and repeated activa-

as cardiomyocytes, lung fibroblasts and pancreatic βtion results in smaller responses with subsequent addition

cells1,3. TRPA1 is activated by a range of natural pungentof the same concentration of ligand (tachyphylaxis)12.

compounds

includingallyl

isothiocyanate(AITC),

cinnamaldehyde, and allicin . TRPA1 can also be
4-6

activated

by

cold

temperature3,7

andhasproposed to act as a mechanosensor .

1.0

been

Not only3

has TRPA1 been proposed to play a role in noci-0.8

ception and certain pain conditions8-10, but has also in
cardiovascular conditions such as atherosclerosis, heart

0.6

TRPA1 channel. For example, TRPA1 has been identified
as a potential target for persistent chronic painful states
including inflammation, neuropathic pain, diabetes,
fibromyalgia, bronchitis, and emphysema . Indeed,
11I/Imax

failure, arrhythmia, vasodilation, and hypertension1. Thus,within drug development, much attention is paid to theCarvacrol
EC50 = 451 µM (n = 361 wells)

0.4

the TRPA1 antagonist GR 17536 from Glenmark showed
efficacy in a Phase IIa proof-of-concept clinical trial for
peripheral diabetic neuropathy11.
The most challenging aspects involved in the screeningof the TRPA1 channel are the channel’s mechanosensitivity, fast desensitization and activity dependenceon intracellular calcium. Here, we present high quality
data with reliable pharmacology on hTRPA1 expressing
CHO cells collected on the SyncroPatch 384. Data ispresented showing activation of the TRPA1 channel bycarvacrol by the antagonists AMG0902 and A967079.
Download more Application Notes from www.nanion.de

100 µM
200 µM
300 µM
400 µM
500 µM
600 µM

0.20.5 nA

0.0
6 7 8 910-423452s
6 7 8 910-3[Carvacrol (M)]

23Figure 1: Activation of TRPA1 by carvacrol recorded on the SyncroPatch384. The concentration response curve for carvacrol for an average of 361
wells is shown with the average traces for each concentration shown in
the inset. Multihole chips (4 holes per well) were used, TRPA1 was recorded
using a constant holding potential of -60 mV, a single concentrationof carvacrol was added to each well and the concentration response
curve calculated across multiple wells.

Application Note
In order to minimize desensitization of the channel and1.0

allow repetitive activation of TRPA1, the LigandPuff
approach of the SyncroPatch 384 was used for theAMG0902
IC50 = 35.9 nM (n = 337 wells)

0.8experiments. A small amount of ligand was used to actiactivation of the current but minimizes desensitization.
Using this approach, TRPA1 could be repetitively
activated by repeated stimulation with 300 µM carvacrol(Fig. 2) allowing inhibitors to be studied.
ANormalized Blockvate TRPA1 with a short wait time of 4 secs to ensure full
0.60 nM
3 nM
10 nM
30 nM
100 nM
300 nM

0.4

0.2

0.2 nA
2s

0.0
10-11

0.5 nA
5s

0-1

10-7

10-6Imax (nA)

A967079 is a selective inhibitor of TRPA1 with an IC50 in thenM range and does not block TRPV1, TRPV2, TRPV3, TRPV4-2

or TRPM8 channels14. In our experiments, hTRPA1 activatedby 300 µM carvacrol was blocked by A967079 with an IC50-3-4

10-910-8[AMG0902 (M)]

Figure 3: Average concentration response curve for AMG0902. A single
concentration of AMG0902 was co-applied with 300 µM carvacrol and theconcentration response curve for an average of 337 wells is shown. Average
traces in different concentrations of AMG0902 are also shown in the inset.
The IC50 for AMG0902 was 35.9 nM (n = 337) giving a success rate of 88% forcompleted experiments.

= 9.8 nM (n = 331 wells; Fig. 4), in good agreement with theliterature value of 51 nM for hTRPA1 activated by 100 µM
0100200

300
400Time (s)500600Figure 2: Repetitive activation of TRPA1 by carvacrol. Using the LigandPuff
technique, TRPA1 could be repetitively activated by 300 µM carvacrol atleast 4 times in the same well with a peak amplitude consistent with priorapplications (<10% change in peak amplitude). Shown are raw current
traces in response to 300 µM carvacrol from an exemplar cell in (A) and thecorresponding time course of the experiment in (B).

Each well received 3 x applications of 300 µM carvacrol followed by co-application of carvacrol and a single
concentration of either AMG0902 or A967079. Figure 3
shows the concentration response curve for AMG0902 foran average of 337 wells. The IC50 for AMG0902 was calcu-AITC using manual patch clamp14.
1.0 A967079
IC50 = 9.8 nM (n = 331 wells)
0.8
Normalized Block

B10-100 nM
6 nM
20 nM
60 nM
200 nM
600 nM

0.6

0.4
0.5 nA

0.22s

lated to be 35.9 nM in good agreement with the literature
value of 68 ± 38 nM for AITC activation of rTRPA1 in a Ca2+
imaging assay13.

0.0

10-11

10-10

10-8
10-9
[A967079 (M)]

10-7

10-6

Figure 4: Average concentration response curve for A967079. A single
concentration of A967079 was co-applied with 300 µM carvacrol and theconcentration response curve for an average of 331 wells is shown. Average
traces in different concentrations of A967079 are also shown in the inset.
The IC50 for A967079 was 9.8 nM (n = 331) giving a success rate of 86% forcompleted experiments.

Download more Application Notes from www.nanion.deApplication Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of raw current tracesof TRPA1 expressed in CHO cells as recorded on one NPC-384 patch clamp chip (4x holes per well). Three hundred and eighty-four small color-coded
pictures as seen in the upper left part display 384 recordings. Wells used for analysis are shown in green and grey wells indicate failed wells based on thequality control criteria. One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show current
traces of hTRPA1 after activation with 300 µM carvacrol (blue trace) and with co-application with AMG0902 at the given concentrations (black trace).

Exp end and chip
unload5 min~10 min

2 min
1 min

Chip load andpriming

Patch clamp experiment: 3 control applications (300 µM carvacrol)
made ~3 mins apart, 1 compound co-application or full activation

Analysis

Figure 6: The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a single point concentration response curve plus
activation of TRPA1 with carvacrol took approximately 17 min.

Download more Application Notes from www.nanion.deApplication Note
Figure 5 shows a screenshot of the SyncroPatch 384

tosoftware during an experiment. A color-coded over-single compound concentration to each well.

ensurestability)andapplication

ofaprocedure

ofview (based on seal resistance in this case) of all 384
wells gives the user a good impression of the suc-Usingcess rate of the experiment. The user can choose

the SyncroPatch 384, TRPA1 could be repetitively

whether to visualize raw traces or online analysis. Inactivated. As expected, hTRPA1 could be activatedthis case, raw current traces are shown, blue traces

by carvacrol11 and inhibited by AMG0902 and A967079

indicate control (300 µM carvacrol) and black traces

inin the presence of AMG0902. An individual well can bevalues similar to those reported in the literature

atheLigandPuff

application

concentration-dependent

mannerwith
13,14.

IC50

highlighted to monitor the progression of the experiment.
The SyncroPatch 384 is a high throughput and highly
In conclusion, hTRPA1 expressed in CHO cells canbe recorded on the SyncroPatch 384 with a goodsuccessrate(>86%).

Theabout

17

activationfortimelineminutes
withcompletedof(start

carvacrol

each–experiments

experimentend)and(repeatedwasincluded
3timesReferences

reliable automated patch clamp device for recording
hTRPA1 currents. User-friendly software, excellent successrates, single additions or multiple additions of compoundto each cell and easy analysis result in reliable highquality data at an increased throughput with an
economical cost per data point.

Methods

1. Wang Z., et al. 2019. Front. Pharmacol. 10: 1253.

Cells

2. Story G.M., et al. 2003. Cell. 112(6): 819-29.

hTRPA1 expressing CHO cells were supplied by Charles

3. Nilius B., et al. 2012. Pflugers Arch. - Eur. J. Physiol.

River, USA.

464:425–458.
4. Bandell M., et al. 2004. Neuron. 41 (6): 849-57.

Cell culture

5. Jordt S.E., et al. 2004. Nature. 427: 260-265.

Cells were cultured and harvested according to

6. Macpherson L.J., et al. 2005. Curr. Biol. 15: 929–934.

Nanion’s standard cell culture protocol for gentle cell

7. Kwan KY., & Corey D.P. 2009. JGP. 133(3): 251–256.

handling .

8. Kremeyer, B., et al. 2010. Neuron. 66: 671-680.
9. Koivisto, A., et al. 2018. Pharmaceuticals 11: 117;

Electrophysiology

doi:10.3390/ph11040117.

Perforated patch clamp recordings were conducted

10. Hiyama H., et al. 2018. Mol. Pain. 14: 1744806918789812.

according to Nanion’s standard procedure for the

11. Skerratt S., 2017. Prog. Med. Chem. 6: 81-115.

SyncroPatch 384 using escin in the intracellular solution. A

12. Xu H., et al. 2006. Nat. Neurosci. 9(5):628-635.

constant holding potential of -60 mV was used and a

13. Lehto, S.G., et al., 2016. Mol. Pain. 12: doi:

small volume of ligand (5 µl) was applied to activate

10.1177/1744806916677761.

TRPA1. Multihole chips were used with 4 holes per well. For

14. Chen, J., et al. 2011. Pain. 152(5): 1165-1172

pharmacology experiments the inhibitor was co-appliedwith activator after 3 control applications of ligand anddata normalized to peak amplitude in the absence ofthe blocker. A single concentration of blocker wasapplied to each well and the concentration response
curve calculated across multiple wells.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960