Channel:
Cells:
Tools:

Application NotehTRPA1
HEK293Port-a-Patch

TRPA1 activation by allyl isothiocyanate
recorded on the Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by EMD Millipore, USA.

Summary

Results

Transient receptor potential (TRP) channels are an

Current responses of a cell expressing hTRPA1 andimportant class of receptors found widely distributed​

activation by AITC are shown in Figure 1.

throughout the mammalian central and peripheral
nervoussystems.

activatedbyTheymany

havestimuli

beenshownincludingtobetemperature,mechano-stimulation, divalent cations and pH, amongst
others. TRP channels are receiving much attention as
potential targets for the treatment of, for example, pain,respiratory diseases such as asthma, cancer and immune
disorders (for review see ref. 1).
The TRPA1 receptor was first cloned from cultured
human lung fibroblasts but has subsequently been foundto be expressed in sensory neurones and is often foundco-localised with TRPV1 (for review see ref. 2). TRPA1 is
activated by a number of chemical stimuli including
allyl isothiocyanate (mustard oil), cinnamaldehyde (theactive ingredient of cinnamon), chlorobenzylidene
malononitrile (CS tear gas), hydrogen peroxide andhyperchlorite (chlorine gas). It is thought that TRPA1,
together with TRPV1, may contribute to chemical
hypersensitivity, chronic cough, and airway inflammationin asthma2.
Here

we

present

data

recordedon

thePort-a-Patch® with external perfusion showing recordings ofhumanTRPA1(hTRPA1)

activatedbyincreasing

concentrations of allyl isothiocyanate (AITC). hTRPA1
could be repetitively stimulated using low concentrationsof AITC but was desensitized by high concentrations ofAITC (>10 µM).

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Figure 1:
The TRPA1 current could be activated by increasing concentrations ofAITC. A Current responses of an exemplar HEK cell stably transfectedwith hTRPA1 to increasing concentrations of AITC (1 - 30 µM). B Timecourse of the experiment. Bars indicate application of AITC at theconcentrations shown. C The concentration response curve for AITC.
EC50 = 2.7 ± 0.4 µM (n = 5), Hill coefficient = 2.5 ± 0.2 µM (n = 5). The EC50
value agrees well with the literature values2,3.

Application NotehTRPA1 was activated by AITC in a concentrationdependent manner. The maximal response was elicitedwith 10 µM. 30 µM elicited a smaller response than 10 µM,
presumably due to receptor desensitization. The EC50 for
AITC was calculated from the concentration response
curve as 2.7 ± 0.4 µM (n = 5). This is in good agreementwith values reported in the literature2,3.
Figure 3:
The hTRPA1 current was desensitized by 10 µM AITC. A hTRPA1 was activated by 2 consecutive applications of 10 µM AITC. In this case, the 2ndapplication of AITC produced about 50% of the current amplitudeachieved with the first, presumably due to receptor desensitization. B Timecourse for the experiment. Bars indicate application of 10 µM AITC.

When a higher concentration of AITC was used (10 µM),
the response to the 2nd application of AITC was approximately 50% of that of the first.
In summary, HEK cells stably transfected with the hTRPA1
Figure 2:
The hTRPA1 current could be repetitively stimulated by 3 µM AITC. A hTRPA1was activated by 3 consecutive applications of 3 µM AITC. Using this concentration of AITC, TRPA1 could be reliably stimulated. B Timecourse for theexperiment. Bars indicate application of 3 µM AITC.

receptor can be captured to the patch clamp aperture of the Port-a-Patch and TRPA1 can be activatedusing AITC giving an EC50 value consistent with those published in the literature. Using a submaximal concentrationof AITC (3 µM), hTRPA1 could be repetitively stimulated
making pharmacological experiments possible. Higher

Using low concentrations of AITC (3 µM), TRPA1 could beconcentrations of AITC caused strong desensitization ofrepetitively stimulated in the same cell. Figure 2 shows thethe channel. The Port-a-Patch provides an excellent tool

current response of hTRPA1 to 3 consecutive applications

for studying the TRPA1 channel and it’s agonists and an-of 3 µM AITC.

tagonists acting both internally and externally.

References

Electrophysiology

1. Clapham, D., 2003. Nature. 426: 517-524
2. Bessec, B.F., Jordt, S-E., 2008. Physiology. 23: 360-370
3. McNamara C.R., et al, 2007. PNAS. 104 (33): 13525 13530

Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for the Porta-Patch. The external perfusion system was also used.
Currents were recorded every 1 s by 200 ms voltage
ramps from -100 mV to +100 mV. Currents were elicited

Methods

by application of AITC via the external perfusion system.

Cells

externalAITC was made as a 10 mM stock in DMSO and diluted inPrecisION hTRPA1-HEK recombinant cell line (CYL3066)

recordingindicated.

was kindly supplied by EMD Millipore, USA.
Cell culture
Cells were cultured and harvested according to Nanion’s standard cell culture protocol.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

solution

tothe

concentrations