Application NoteChannel:
TRPV1Cells:
Bilayers
Tools: Orbit Mini, Temperature
ControlTemperature activation of TRPV1
reconstituted into planar lipid bilayers
and recorded using the Orbit mini
platform
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

The transient receptor potential cation channel, subfam-

Formation of planar lipid bilayers on the MECA4

ily V, member 1 (TRPV1), is a ligand-gated, non-selective

The bilayers are painted over the 4 wells of the MECA4

cation channel widely expressed in the peripheral andchip using either a hair brush or a glass rod. The lipid used

central nervous system. The TRPV1 channel can be ac-in this study was DPhPC (1 mg/ml) dissolved in Octane.

tivated by a number of physical and chemical stimuli,

The brush or the glass rod was dipped into the lipids be-

including capsaicin (the active ingredient in chili pep-fore painting over the wells.

pers), noxious heat (typically >42˚C) and low pH. The

Once the bilayers were formed, the purified protein

TRPV1 channel is putatively involved in the perception

TRPV1 was added to the bath solution for the reconstitu-and transmission of painful stimuli. Importantly, this chan-tion into bilayers. The activity of TRPV1 was checked usingnel is proposed to underlie many chronic pain states

the agonist, Capsaicin (fig.1). The capsaicin was washedincluding inflammation, neuropathic pain and cancer

away before starting applying elevated temperature.

pain, amongst others (1). These types of pain states are
currently poorly managed by the pain medications available and this has led the pharmaceutical industry to seeknovel targets for pain management, such as TRPV1. However, TRPV1 antagonists have so far failed in clinical trials
due to an undesirable increase in core body temperature(2) resulting in hyperthermia. From these studies, it is proposed that tonically active TRPV1 channels are involvedin maintaining normal body temperature and this could
have significant influences on drug design. Finding novelcompounds with differing effects on capsaicin activationand heat activation may be crucial in the discovery oflead compounds for the treatment of pain and other disease states.
Here we present data collected on the 4-channel Orbitmini with temperature control showing the potential useof the Orbit mini to record heat activation of single channel TRPV1.

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Figure 1. Current traces of reconstituted TRPV1 in DPhPC bilayers

recorded at a transmembrane voltage of Vm= +50 mV. TRPV1
is activated using 100 μM Capsaicin (at room temperature). The
recordings were done in symmetrical 200 mM KCl, 10 mM HEPES, pH
7.2 (with 20 kHz sampling rate and 10 kHz bandwidth).

Application Note

The temperature was raised progressively from Roomtemperature (~25 degrees Celsius) to 60 degrees Celsius.
The figure 2 shows the current traces of bilayers with TRPV1
reconstituted at different temperatures.

Figure 2. Current traces of reconstituted TRPV1 in DPhPC bilayers

recorded at a transmembrane voltage of Vm= +50 mV. The activityof TRPV1 is temperature dependent. The activity of TRPV1 at 3
different temperatures around the activation threshold (>42 oC) 25,
35 and 50 oC is shown. The recordings were done in symmetrical 200
mM KCl, 10 mM HEPES, pH 7.2.

Figure 4. Effect of temperature on TRPV1 activity. Top, the openprobability (Po) versus the temperature and below,the Arrhenius
plot of the same data resulting in a Q10 of 72

The conductance was derived from all point histograms
and was found to be similar to previously published dataon single channel conductance of TRPV1 in potassium solution (1). The conductance increased slightly with raisingtemperature (figure 4) as expected.
Figure 3. Current trace Zoomed in of reconstituted TRPV1 in DPhPC

bilayers recorded at a transmembrane voltage of Vm= +50 mV.

Download more Application Notes from www.nanion.deApplication Note
The open probability was determined for each tempera-

in parallel were recorded giving better statistics ofture together with the conductance of the channel

the TRPV1 activity. The temperature range from 20 toopening. Figure 3 shows the open probability (Po) versus

60˚C was applied using our Temperature Control system

the temperature. This graph allows us to show the Arrhe-connected to the Orbit mini.

nius plots (right) of the same data with Po values as thenatural logarithm (ln) and temperature (T) as reciprocalKelvin. All single channel currents were recorded ina symmetrical K+ solution. The open probability of TRPV1
increases with increasing temperature as shown in figure
3 (left). Bilayers without proteins did not respond to varying temperatures. Po values were obtained for temperatures between 25 and 60 ˚C and were used to calculate
the Q10 values of 72 from the Arrhenius plot of TRPV1. The
heat activation of TRPV1 was reversible. The active cooling of the temperature control of the Orbit mini allowed
us to cool down rapidly from 60 to 20 ˚C.
In conclusion, we have demonstrated stable recordingsof the TRPV1 channel on our Orbit mini platform. 4 channels

Figure 3. Temperature dependance conductance of TRPV1. Theconductance increase slightly due to the raise of the temperature.
Above 60 oC, the conductions was not calculated due to possible
evaporation.

References

Methods

1. Mandadi, S., Roufogalis, B.D. 2008. ThermoTRP Chan-

Bilayer formation and recordings

nels in Nociceptors: Taking a lead from Capsaicin recep-

Symmetrical solutions were used: 200 mM KCl, 10 mM

tor TRPV1. Curr. Neuropharmacol. 6:21-38

HEPES, pH 7.2.

2. Gavva, N.R., Treanor, J.J, Garami, A., Fang, L., Surapaneni, S., Akrami, A., Alvarez, F., Bak, A., Darling, M., Gore,The Orbit mini has an integrated 4 channel amplifierA., Jang, G.R., Kesslak, J.P., Ni, L., Norman, M.H., Palluco-(eFOUR from Elements, Cesena, Italy) and integrateni, G., Rose, M.J., Salfi, M., Tan, E., Romanovsky, A.A., Ban-the MECA4 chip, multi-electrode-cavity-array chips

field, C., Davar, G. 2008. Pharmacological blockade of(IONERA, Freiburg, Germany). Bilayers are formed by

the vanilloid receptor TRPV1 elicits marked hyperthermia

painting 1,2-diphytanoyl-sn-glycero-3-in humans. Pain. 136: 202-210.

phosphocholine (DPhPC, Avanti Polar Lipids)

3. Damien S.K. Samways and Terrance M. Egan. Calcium-dissolved in octane (1 mg/ml) over the 50 μm

dependent decrease in the single channel conductancediameters wells of the MECA4. Recordings wereof TRPV1. Pflugers Arch. 2011 Nov; 462(5): 681–691

done at 10 kHz bandwidth and 20 kHz sampling rate.
The purified protein, TRPV1 in detergent (DDM, 0.1%),
with a concentration of 150 ng/ml, was added to thebath solution after the formation of the bilayers at anapplied potential of +50 mV.

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