Application NoteChannel:
TRPM8, TRPV1Cells:
HEK293Tools:
Port-a-Patch
External Perfusion System

TRPV1 and TRPM8 recorded on
Nanion's Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.
Data was kindly provided by Dr. D. Cohen, Oregon University, Portland, USA.
SummaryResults

TRPV1 and TRPM8 are members of the transient receptor

Whole cell current responses of an individual cell expres-potential channel (TRP) family. This family was designatedsing the TRPV1 channel is shown in Figure 1. The activator

TRP because of a spontaneously occurring Drosophila

was applied and washed out using Nanion´s External Per-mutant lacking TRP that responded to a continuous lightfusion System.

with a transient receptor potential.

8I (pA)

2 µM Capsaicin

TRPV1 is mainly expressed in sensory nerves. Its presenceis essential for transduction of nociception as well as inflammatory and hypothermic effects of vanilloid com-

6pounds. It also contributes to acute thermal nociception
and thermal hyperalgesia following tissue injury.
4TRPV1 forms a relatively Ca2+-selective ion channel withoutwardly rectifying properties. It is activated by vanilloids such as capsaicin, by protons, increased tempe-2ratures, lipoxygenase products, as well as anandamidecontrol(Huang et al., 2002).
Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food andpharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset ofsensory neurons from dorsal root and trigeminal ganglia,
due to activation of TRPM8, a Ca2+-permeable, cold-activated member of the TRP superfamily of cation channels(McKemy et al., 2002; Peier et al., 2002).
Here we present data of TRPV1 and TRPM8 collected on
the Port-a-Patch. Channel activation with capsaicin as
well as menthol are shown.

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washout

V (mV)-100
Figure 1:-50

50

100

Whole cell current responses from HEK293 cells transiently expressing TRPV1 to a ramp voltage protocol (-100 mV to +100mV). 2 µM capsaicin reversibly activated the channel. Data
were kindly provided by Dr. David Cohen, Oregon Health &Science University, Portland, USA.

Application Note500 pA

100 msFigure 2 shows representative traces from an individual cellstably expressing TRPM8 in the absence and presence ofincreasing concentrations of menthol. The activator wasapplied using the Perfusion System. The exchange times

1.5

for a full solution exchange using the Perfusion System are

300 µM Menthol

I (nA)

approx. 150 ms. A full concentration response of menthol
ranging from 1 µM to 300 µM was achieved.
control
1 µM Menthol
10 µM Menthol
30 µM Menthol
100 µM Menthol
300 µM Menthol
wash1.0

1.0

I (nA)0.50.5-60

V (mV)-60-40washout

V (mV)-20

204060Figure 2:
Increasing concentrations of menthol acting on the TRPM8 channel. Afterthe highest concentration was applied, the activator was washed outagain. Ramp voltage protocol was applied from -70 mV to +70 mV within
200 ms.

-40-20204060Figure 3:
Current-voltage relation of TRPM8 expressing cells in the presence of 300µM menthol (top, left) and after washout (top, right). Corresponding IVcurves of the steady state current (below). Voltages were applied from-60 mV to +60 mV for 500 ms.

-60 mV up to +60 mV. Recordings were performed in thepresence of 300 µM menthol and after washout (Figure 3).
In conclusion, we have demonstrated stable recordings

Another example shows the time-dependent increase ofof the TRP channels TRPV1 and TRPM8 on a planar patch

whole cell currents when applying 500 ms pulses fromclamp system.

References

Methods

1. Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De Pe-Cells

trocellis L, Fezza F, Tognetto M, Petros TJ, Krey JF, Chu CJ,Miller JD, Davies SN, Geppetti P, Walker JM, Di Marzo V(2002). „An endogenous capsaicin-like substance withhigh potency at recombinant and native vanilloid VR1
receptors“. Proc. Natl. Acad. Sci. U.S.A. 99 (12): 8400-5.

HEK293 cells stably expressing TRPM8 (Millipore) wereused.

HEK293 cells transiently transfected with TRPV1

were kindly provided by Dr. David Cohen, Oregon Health& Science University, Portland, USA.
Cell culture

2. McKemy DD, Neuhausser WM, Julius D (2002) Identifi-Cells were cultured and harvested according to Nanion‘scation of a cold receptor reveals a general role for TRP

standard cell culture protocol.

channels in thermosensation. Nature 416: 52-58.

Electrophysiology

3. Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, Earley TJ, Dragoni I, McIntyre P,
Bevan S, Patapoutian A (2002) A TRP channel that senses
cold stimuli and menthol. Cell 108: 705-715.

Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for the Porta-Patch. For the application and washout of the activators, Nanion´s External Perfusion System was used.

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