Application Note

Channel:Cells:
Tools:hTRPM8CHOPatchliner®

TRPM8 activation by menthol and Eucalyptol
performed on Nanion’s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Chantest.

SummaryResults

TRPM8 is a member of the transient receptor potential

For

channel (TRP) family. TRPM8 is known to be a thermo-Patchliner of the hTRPM8 (CHO) cells, success rate (cells

sensitive channel, activated by cold temperaturessealed), seal resistance (RSeal) and average current in(below ~25˚C) and ligands such as menthol, Eucalyptol

the presence of agonist were determined (Table 1).

theevaluationoftheperformanceonthe®

and icilin. It belongs to the melastatin subfamily of TRP
channels1 and shows an outward rectification with a

The whole cell current of an individual cell expressing

relatively high permeability for calcium ions andTRPM8 channel is shown in Figure 1A. The recording shows

little selectivity between monovalent cations. Menthol,

the activation of the TRPM8 by menthol (1 mM) followed

a secondary alcohol produced by the peppermintby wash using Nanion’s standard external solution. The

herb, Mentha piperita, is widely used in the food andcorresponding time course of the experiment is shown

pharmaceuticalin Figure 1B. The current elicited by menthol was fullyasa

cooling/soothing

compound and odorant. It induces Ca

2+

influx in a

subset of sensory neurons from dorsal root and trigeminal
ganglia, where the TRPM8 channel is specificallyreversible upon washout.

Aexpressed2,3.

control
1mM Menthol
wash

Here we present data of hTRPM8 collected on thePatchliner . Cells performed well on the Patchliner®

B

Channel activation by the agonists menthol andEucalyptol is shown.
RSeal (GΩ)

Whole cell

Current
amplitude(pA)

85/96 (89%)

1.6 ± 2.2

79/85 (93%)

876 ± 358

Table 1:
Seal parameters for CHO cells expressing hTRPM8 recorded on thePatchliner®. Cells sealed with a high success rate (89%) with an averageseal resistance (RSeal) > 1 GΩ.

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1.5

Wash

0.500.5 nA
50 mV

Cells sealedMenthol
1 mM

2.01.0®

with a success rate of 89% for seal resistance >600 MΩ.

2.5

current (nA)industries

0.0
20 s

Figure 1:
A Whole cell current responses from induced CHO cells expressing TRPM8 toa ramp protocol (-80 mV to +80 mV over 200 ms) in the presence, absence
and after wash of 1 mM menthol. The holding potential was set at -40 mV toeliminate any possible contributions by voltage-dependent sodium channel
activity. B Time course measurement of TRPM8 activation with menthol

Application NoteThe concentration response relationship obtained forconcentration (1 mM) on the same cell is also shown inmenthol using a voltage ramp protocol is shown in Figure

Figure 3.

2. The EC50 = 148 ± 16 µM (n = 7; Hill coefficient 1.85). This isA

in good agreement with the literature .
4,5control
1 mM Eucalyptol080

0.5 nA60

B

402050 mV
3.0
2.5

10-6Menthol 1mM

current (nA)

% Activation

100

2.0

10-5
10-4
10-3
[menthol] (M)Eucalyptol 1mM

1.5Figure 2:
Concentration response curve for menthol with representative Hill fit. TheEC50 for menthol was 148 ± 16 µM (n = 7).

1.0
0.5
0.020 sAnother molecule which is known to activate TRPM8is Eucalyptol, an essential oil often used in alternative
medicine in analgesic and anti-inflammatory remedies.
The effect of Eucalyptol on TRPM8 activation was lessthan that of menthol. TRPM8 activation by Eucalyptol
is shown in Figure 3. Whole cell current responses frominduced CHO cells expressing TRPM8 to a ramp
protocol by 1 mM Eucalyptol followed by a wash areshown. The time course of the experiment showing
activation by Eucalyptol and menthol at the same

Figure 3:
A Whole cell current responses from induced CHO cells expressing TRPM8 toa ramp protocol (-80 mV to +80 mV over 200 ms). 1 mM Eucalyptol activatedthe channel. B Time course of the experiment. Activation of Eucalyptol andmenthol on the same cell. Eucalyptol activated TRPM8 to a lesser degreecompared with menthol activation.

In conclusion, hTRPM8 expressed in an inducible CHO
cell line provided by Chantest can be reliably recordedon the Patchliner® with activation by menthol4,5 andEucalyptol as expected.
expression was induced by the addition of 1 mg/ml

References

tetracycline at least 24 hours prior to measurements.
Patchliner® recordings were performed 24 to 36 hours

1. Montell, C., et al., 2002. Cell 108: 595–598.

after induction. Induction time of shorter than 24h, or

2. McKemy, D.D., et al., 2002. Nature 416: 52-58.

longer than 36h, led to a lower success rate in terms of

3. Peier, A.M., et al., 2002. Cell 108: 705-715
4. Andersson, D.A., et al., 2004. J. Neurosci. 24(23):5364–currents observed and seal rate, respectively.
Electrophysiology

5369
5. Zhang, X., et al.,2012. Nat. Cell Biol. 14(8):850-858

Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theMethods

Patchliner®. Currents were elicited by 200 ms voltage
ramps from -100 mV to +100 mV, Vhold = -40 mV. ForCells
CHO cells stably expressing hTRPM8 provided byChantest.

agonist activation of channels, menthol and Eucalyptol
were diluted in external solution at the indicatedconcentrations and applied at room temperature for

Cell culture

approximately 40 s before wash with external solution.

CHO cells stably expressing hTRPM8 under tetracycline
induction control were grown at 37°C, 5% CO2 in ahumidified

atmosphere

intheincubator.

hTRPM8Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax80636 Munich, Germany

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