Application NoteChannel:
hTRPA1
Cells:
HEK293Tools:
SyncroPatch® 384PE

High Throughput Activation and Block ofhTRPA1 on Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were supplied by EMD Millipore, USA

Summary

Results

Transient receptor potential (TRP) channels have become

For the evaluation of the performance of hTRPA1(HEK293)

important potential targets in drug discovery for the treat-cells, Seal Resistance, Cslow and the Series Resistancement of, for example, pain, respiratory diseases such as(Rseries) were determined from one experiment (Fig. 1) .

asthma, cancer and immune disorders, multiple kidney

AThe transient receptor potential ankyrin 1 (TRPA1), a
member of the TRP family of cation channels, plays
a predominant role in the sensation of noxious cold²

40
20light TRPA1 antagonists as a promising new approachfor the treatment of acute and chronic pain. Indeed, a
TRPA1 antagonist has shown positive results in a proof-ofconcept study for diabetic neuropathic pain6.
The most challenging aspects involved in the screening of the TRPA1 channel are the channel’s mechanosensitivity, fast desensitization and activity dependence
on intracellular calcium. Here, we present high quality
data with reliable pharmacology on hTRPA1 expressing
HEK cells collected on the SyncroPatch® 384PE. Data ispresented showing activation of the TRPA1 channel by
SCMA and inhibition by A-967079.
Download more Application Notes from www.nanion.de0Cslow (pF)ΩG

200-30and data from a recent human genetic study5 high-20-25

attention is paid to the TRPA1 channel. Preclinical data404025

painful responses². Thus, within drug development, much

60600110 0-220 0-330 0-440 0-50(DRG) and trigeminal ganglion, both of which transmit

Cells (%)

expressed in sensory neurons of the dorsal root ganglion

C2080-20B

function in nociception, TRPA1 has been shown to be

15

proposed

-1
5its

10

with510Consistent0-stress4.

Seal Resistance

5-cinnamon, as well as metabolites produced during

Cells (%)

dicompounds found in foods such as garlic, mustard and>1sablrange of environmental irritants causing pain, pungented
<100
10M0
Ω
-500
50
M0Ω
M
Ω
-1
G
Ω

0and inflammatory pain3. The channel is activated by aoxidativestart of experimentend of experiment60
Cells (%)

diseases and skeletal disorders1.

Rseries (MΩ)

Figure 1: Statistics of TRPA1 expressing HEK293 cells recorded on one
NPC384 chip on the SyncroPatch® 384 PE
A Success rate (seal resistance) of individual HEK 293 cells on theSyncroPatch® 384. Shown is a bar graph of seal resistances at the start(light blue) and end of the experiment (dark blue). B Bar graph of cell
capacitance (Cslow) and C Series Resistance (Rseries) values for HEK293
cells expressing TRPA1.

Application Note
Currents mediated by hTRPA1 could be reliably andsustainably activated by the agonist Supercinnamaldehyde (SCMA; 5 µM) and blocked by A-967079 in aconcentration-dependent

manner(Fig.

2).

TRPA1

currents were first activated by SCMA (white region), a
single concentration of A-967079 was applied to eachcell (blue region) and finally a high concentration ofA-967079 (grey region) was applied to fully block thecurrent.
Figure 3: Average concentration response curve for A-967079
The concentration response curve was constructed across the whole plate.
Only cells which returned to baseline after the full block concentration ofA-967079 were used for analysis giving an IC50 = 6.6 nM (n = 242) and a
success rate of 63% for completed experiments.

TRPA1 has been shown to be mechanosensitive8 and weobserved activation of TRPA1 when control solution wasadded to the cells at a speed of 5 µl/s (Fig. 4). This mechanical activation was also observed during application ofantagonist. To avoid this mechanical activation of TRPA1
during activation with SCMA the solution was added usinga very slow speed (2.5 µl/s) and the travelling speed of theFigure 2: Activation of TRPA1 by SCMA and concentration-dependent blockby A-967079
Ramp recordings (left) and snapshots of the corresponding time course of thecurrent amplitude at 60 mV (right) of exemplar HEK cells expressing hTRPA1.
A Current responses after exposure to 5 µM SCMA (left panel: black trace;right panel: white region), block by 15 nM A-967079 (left panel: light bluetrace; right panel: blue region)) and full block by 5 µM A-967079 (left panel:dark blue trace; right panel: grey region). B Current responses after exposure
to 5 µM SCMA (left panel: black trace; right panel: white region), block by
500 nM A-967079 (left panel: light blue trace; right panel: blue region)) andfull block by 5 µM A-967079 (left panel: dark blue trace; right panel: greyregion).

pipette was also adjusted to minimise pressure changes atthe cell when the pipette enters the well.

Each cell was exposed to a single concentrationof A-967079 and the concentration response curve
calculated across the whole plate. The average
concentration response curve for 242 cells is shown in
Figure 3. The calculated IC50 was 6.6 nM (n = 242) which

is similar to that reported in the literature (67 nM)7 . Only
cells which returned to the baseline (recorded at thestart of the experiment) upon application of the full-block
concentration of A-967079 (5 µM) were included in theFigure 4: TRPA1 is also mechanosensitive.
Screenshot of the PatchControl 384 software during an experiment activating
TRPA1 using control solution added at a speed of 5 µl/s. This effect was notblocked by A-967079 (blue region).

analysis. In this experiment, 242 cells were used for theconcentration response curve giving a success rate of
63% for completed experiments.

Nanion Technologies GmbHphone +
 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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

Application NoteFigure 5:
Graphical user interface of the screening and data analysis software used on the SyncroPatch® 384PE. Screenshot of depiction of online analysis data ofhTRPA1 expressing HEK293 cells as recorded on one NPC-384 patch clamp chip. Three hundred and eighty-four small color-coded pictures as seen in theupper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 500 MΩ), blue (Rmemb = 100–500 MΩ), light blueor grey (Rmemb < 100 MΩ or cells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right.
Graphs show current amplitudes of SCMA (5 µM) activation (white region), block by A-967079 (blue region) at the indicated concentrations and full block(grey region) using 5 µM A-967079. One minute of baseline current was recorded prior to application of SCMA. Only cells which returned to baseline after
the full block concentration of A-967079 were used for analysis.

Exp end and chip
unload
6 - 8 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash + 3 compound
additions (activation, block with compound of interest, fullblock)

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 SCMA and full block at the end of the experiment took approximately 13-15 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax

80636 Munich, Germany

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

Application Note
Figure 5 shows a screenshot of the SyncroPatch® 384reducing the consumable cost per data point tosoftware during an experiment. A color-coded overview< $0.60.

(based on seal resistance in this case) of all 384 wells
gives the user a good impression of the success rate of

The activation properties of hTRPA1 recorded on thethe experiment. The user can choose whether to visualize

SyncroPatch® 384PE are in excellent agreement with

raw traces or online analysis. In this case, online analysis

those reported in the literature9. As expected, hTRPA1

is chosen and the graphs represent current amplitude

could be activated by SCMA10 and inhibited by A-967079

plotted against time. An individual well can bein a concentration-dependent manner with an IC50

highlighted to monitor the progression of the experiment.

similar to that reported in the literature7. As has been pre-In the Online Analysis view, the time points at which

viously reported8, we found that TRPA1 was mechanosolution additions have been made are indicated bysensitive and could be activated by pipetting control

vertical lines, as well as different background colors. Insolution at a speed of 5 µl/s. However, due to the flexibility

this case, white shows activation of TRPA1 by SCMA, blueof the experimental settings of the SyncroPatch® 384PE,

shows inhibition by the indicated concentration of thethe application speed and the travelling speed of theTRPA1 antagonist A-967079 and grey shows full block by

pipette can be adjusted to ensure that the mechanical

5 µM A-967079.

stimulation is eliminated.

In conclusion, hTRPA1 expressed in HEK293 cells canbe recorded on the SyncroPatch® 384PE with a goodsuccess rate. The timeline of each experiment was
about 13-15 minutes (start – end) and included wash,
activation with SCMA, time to reach a stable current,
single compound concentration application and fullblock, all performed on each individual cell, drastically

References

The SyncroPatch® 384PE is a high throughput and highly
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. Clapham, D. 2003. Nature. 426: 517-524
2. Story G.M., et al. 2003. Cell. 112(6): 819-29.

Cells

3. Bautista D., et al. 2006. Cell. 124: 1269–1282

PrecisION - hTRPA1 expressing HEK293 cells (Item Number

4. Bautista D., et al., 2005. Natl. Acad. Sci. U.S.A.,102 (34):CYL3066) were supplied by EMD Millipore, USA.

12248-52
5. Kremeyer, B., et al. 2010. Neuron. 66: 671-680

Cell culture

6.http://www.prnewswire.com/news-releases/glen-Cells were cultured and harvested according to Nan-marks-trpa1-antagonist-grc-17536-shows-positive-data-

ion’s standard cell culture protocol for gentle cell han-in-a-proof-of-concept-study-275445961.html

dling .

7. Chen, J., et al. 2011. Pain. 152(5): 1165-72
8. Brierley, S.M., et al. 2011. J. Physiol. 589(14): 3575-93

Electrophysiology

9. Bandell, M., et al. 2004. Neuron. 41 (6): 849-57

Whole cell patch clamp recordings were conducted

10. Macpherson, M.J., et al. 2007. Nature. 445: 541-45

according to Nanion’s standard procedure for theSyncroPatch® 384PE. A voltage ramp protocol from -50
mV to +60 mV in 60 ms was applied to the cells every 2 s.
Current amplitude at 60 mV was used for analysis.
Solutions were applied to the cell at a speed of 2.5 µl/s.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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