Application NoteChannel:
Cells:
Tools:hTRPM8CHO
Port-a-Patch

Cold activation of TRPM8 using the External
Perfusion System and the Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

TRPM8 is a member of the transient receptor potential

TRPM8 can be activated by a number of ligands

channel (TRP) family. TRPM8 is known to be a thermo-including the cooling compounds menthol and icilin1-4,

sensitive channel, activated by cold temperatures

as well as cold temperatures1-4.

(below ~25˚C) and ligands such as menthol, Eucalyp-channel

tol and icilin1-4. It belongs to the melastatin subfamily ofexhibiting a strong outward rectification at depolarized

TRP channels5 and shows an outward rectification with amembrane potentials and fast, voltage-dependent

relativelyandclosure at negative potentials9,10. TRPM8 -mediatedlittle selectivity between monovalent cations. Menthol,currents are suppressed by high temperatures2,10. Figure

a secondary alcohol produced by the peppermint

1 shows activation of TRPM8 expressed in CHO cells byherb, Mentha piperita, is widely used in the food andcontinuous perfusion of external solution at 10°C and sub-pharmaceuticalsequent suppression by elevated temperature (35°C).

highpermeability

industries

foras

calciumaions

cooling/soothing

compound and odorant. It induces Ca

influx in a2+

subset of sensory neurons from dorsal root and trigeminal

Awith

TRPM8 is a cation

voltage-dependenceofactivation

10°C
35°C

ganglia, where the TRPM8 channel is specifically
expressed2-4. It is known that TRPM8 plays a role cold
allodynia

after

inflammation5andit

has

been

implicated as a possible pharmaceutical target totreat chronic pain and migraine . Additionally, TRPM8cancer7,8, in particular prostate cancer7 and pancreaticcancer8, and may provide novel clinical biomarkers andtherapeutic targets for these types of cancer.
Here we present data of hTRPM8 collected on thePort-a-Patch using cooled solution via the External
Perfusion System. Cold activated hTRPM8 was blockedby capsazepine with an IC50 in good agreement with theliterature .
1,9Download more Application Notes from www.nanion.de50 msB
5Amplitude (nA)channels have been implicated to play a role in

1 nA0 nA

610°C

435°C10°C

32
10100

150

200250300Time (s)

350

400

450

Figure 1: A Whole cell current responses from induced CHO cells expressing
TRPM8 to a ramp protocol (-100 mV to +100 mV over 200 ms) at 10°C (darkblue) and 35°C (light blue). TRPM8 was activated at 10°C but not at 35°C.
B Timecourse of TRPM8 activation with cold solution (10°C) and block bywarm solution (35°C).

Application Note
TRPM8 was activated by cooled solution and this could

A concentration response curve for capsazepine using

be blocked by either increasing temperature, or bycooled solution (10°C) as the activator could also beapplying the blocker capsazepine via the external solution.

constructed. Increasing concentrations of capsazepine

Capsazepine was applied at 10°C. Figure 2 shows theat 10°C were applied and the concentration responsetime course of the exeriment and corresponding example

curve is shown in Figure 3. The IC50 for capsazepine blocktraces.

the literature1,9.

10°C
35°C
50 µM capsazepine (10°C)
Washout (10°C)
1 nA20 ms0 nA

BWashout
50 µM
capsazepine
10°C

35°C

Amplitude (nA)

510°CCapsazepine
IC50 = 12.9 µM

1.0Normalized Ipeak

Aof TRPM8 was 12.9 µM (n = 1), in excellent agreement with

0.8
0.6
0.4
0.2
0.0

3454678 92

10[Capsazepine (µM)]

345Figure 3: Concentration response curve for capsazepine using cooled
solution to activate TRPM8 reveals an IC50 of 12.9 µM, in good agreementwith the literature1,9.

3
2In conclusion, hTRPM8 expressed in CHO cells can be1

reliably activated using the External Perfusion System

00200

400Time (s)600800Figure 2: A Traces from an example cell showing activation by cooledsolution (10°C, dark blue), block by either heated solution (35°C; lightblue), or capsazepine (50 µM; red trace) and washout after capsazepine
application (grey). TRPM8 current could be almost completely recovered
upon washout of capsazepine. B Timecourse of the experiment.

of the Port-a-Patch with Temperature Control whichenables cooling of the external solution to 10°C. Coldactivated TRPM8 was also blocked by capsazepine as
expected1,9. The Port-a-Patch is therefore a useful tool for
identifying TRPM8 inhibitors as potential therapeutics forchronic pain and some cancers.

References

Cell culture

1. Behrendt, H.J., et al., 2004. Br. J. Pharmacol. 141, 737–

CHO cells stably expressing hTRPM8 were cultured using

745

standard culture conditions for the Port-a-Patch and

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

induced using 1 mg/ml tetracycline for at least 24-36

3. Peier, A.M., et al., 2002. Cell 108: 705-715

hours prior to measurements.

4. Andersson, D.A., et al., 2004. J. Neurosci. 24(23):5364–
Electrophysiology

5369
5. Weyer, A.D. & Leyto, S.G. 2017. Pharmaceuticals. 10: 37
6. González-Muñiz, R., et al. 2019. Int. J. Mol. Sci. 20: 2618
7. Hantute-Ghesquier, A. et al. 2018. Pharmaceuticals. 11:58
8.Yee, N.S. et al., 2012. Scientifica. 2012: Article 415158
9. Malkia, A., et al., 2009. Mol. Pain. 6: 62
10. Nilius, B., et al. 2005. J. Physiol. 567.1: 35–44

Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for the Porta-Patch. Currents were elicited by 200 ms voltage ramps
from -100 mV to +100 mV, Vhold = -80 mV. External
solution was cooled to 10°C using the Temperature
Control of the External Perfusion System to activate
hTRPM8. Capsazepine concentrations were made up in
external solution and applied to the cell at 10°C using theMethods

External Perfusion System of the Port-a-Patch.

Cells
CHO cells stably expressing hTRPM8
Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70Afax80339 Munich, Germanywww.nanion.de • info@nanion.de+49 89 218997960