Application NoteChannel:
Cells:
Tools:hTRPV1CHOPatchliner

Heat activation of TRPV1 on Nanion´sPatchliner
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

The transient receptor potential cation channel, subfamily

Current responses of an individual cell to 200 ms voltage

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

ramps (-100 mV to +100 mV) and activation by ­application

cation channel widely expressed in the peripheral­of external solution at increasing t­ emperatures are shownand central nervous system. The TRPV1 channel canin Figure 1. The solution was heated in the ­pipette of thebe ­activated by a number of physical and chemical

Patchliner and rapidly applied to the cell at a speed ofstimuli, including capsaicin (the active ingredient in chili

10 µl/s. A temperature response curve could be con-peppers), noxious heat (typically >42˚C) and low pH. The

structed. Activation began at approximately 60˚C*. The

TRPV1 channel is putatively involved in the perception

EC50 for temperature was estimated to be a­ pproximately64˚C*.

channel is proposed to underlie many chronic pain states

Control RT
55ºC
60ºC
65ºC
70ºC
75ºC

including inflammation, neuropathic pain and cancer
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 seek novel 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. Fromthese studies, it is proposed that tonically active TRPV1channels are involved in maintaining normal bodytemperature and this could have significant influenceson drug design. Finding novel compounds with differing
effects on capsaicin activation and heat activation may
be crucial in the discovery of lead compounds for thetreatment of pain and other disease states.
Here we present data collected on the 4-channel­Patchliner with temperature control showing the ­potential
use of the Patchliner to record capsaicin and heat activation of TRPV1 channels and subsequent block by ruthenium red (RR).

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1.00.8I/Imaxand transmission of painful stimuli. Importantly, this0 nA

0.6
0.4
0.2
0.05 nA
50 ms55

60
6570Temperature (ºC)

Figure 1:
Activation of TRPV1 by increasing temperature. The solution inside the­pipette was heated to the temperature indicated and applied to thecell. TRPV1 was activated at temperatures above 60˚C*. A ­temperature
response plot was constructed and a temperature EC50 of ­approximately
64˚C* was estimated.

75

Figure 2 shows the activation of TRPV1 channels using
either heat (65˚C* or 70˚C*) or capsaicin in the same cell.

AControl RT
Control 60ºC
0.1 µM RR (60ºC)
1 µM RR (60ºC)
10 µM RR (60ºC)
30 µM RR (60ºC)

Following challenge by application of external solution at
65˚C* or 70˚C*, capsaicin (1 µM) was applied at room
temperature.

AControl RT
1 µM Capsaicin (RT)
65ºC

B

0B

0.80 nA

0.6
0.4
0.2

00.12 nA
Control RT
1 µM Capsaicin (RT)
70ºC

HeatCapsaicin

1.0I/Imax

Application Note

50 ms

0.01 0.1110100[Ruthenium Red (µM)]

Figure 3:
A TRPV1 block at 60˚C by RR. RR at increasing concentrations (0.1 µM 30 µM) was pre-incubated and then applied at 60˚C. B IC50 = 7.1 ± 1.2µM (n = 3-6 per concentration); IC50 of capsaicin response by RR was 1.5± 0.2 µM; n = 3.

In summary, TRPV1 receptors stably expressed in CHO

2 nA

5 nA
50 ms

50 ms
Figure 2:
Activation of TRPV1 by capsaicin and temperature in the same cell. TRPV1
could be activated by application of either 1 µM capsaicin or externalsolution heated to A 65˚C* or B 70˚C*.

A full concentration response curve to RR at 0.1, 1, 10and 30 µM could be performed (Fig. 3). RR was first preincubated and then co-applied with heat (60˚C). A
concentration response curve was constructed andthe IC50 was estimated to be 7.1 ± 1.2 µM (n = 3 - 6 perconcentration). The concentration response curve of RR
for capsaicin activation is also shown for comparison (IC501.5 ± 0.2 µM; n = 3).

cells can be reliably activated by noxious temperature(>60˚C*) using the temperature regulation feature ofthe Patchliner. Solution can be heated to temperatures
up to 80˚C* in the pipette and rapidly applied to thecell, transiently activating TRPV1 channels. Additionally,a concentration response curve to the TRP channel
antagonist ruthenium red is shown here. The ability toseparate antagonistic responses of capsaicin and heat
activation of TRPV1 may be critical in novel lead compound
discovery, and therefore, the Patchliner provides a viable,higher throughput alternative to conventional patch
clamp for the discovery of active TRPV1 lead compoundswith a suitable drug profile.

References

Electrophysiology

1. Mandadi, S., Roufogalis, B.D. 2008. „ThermoTRP Channels in Nociceptors: Taking a Lead from Capsaicin Receptor TRPV1.“ Curr. Neuropharmacol. 6: 21-38.
2. Gavva, N.R., Treanor, J.J., Garami, A., Fang, L., Surapaneni, S., Akrami, A., Alvarez, F., Bak, A., Darling, M., Gore,A., Jang, G.R., Kesslak, J.P., Ni, L., Norman, M.H., Palluconi, G., Rose, M.J., Salfi, M., Tan, E., Romanovsky, A.A., Banfield, C., Davar, G. 2008. „Pharmacological blockade of
the vanilloid receptor TRPV1 elicits marked hyperthermia
in humans.“ Pain. 136: 202-210

Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for thePatchliner. Currents were elicited every second by 200 ms
voltage ramps from -100 mV - +100 mV, Vhold = -100 mV.

For heat activation of channels, external solution washeated in the pipette for 90 s and rapidly applied to thecell at a speed of 10 µl/s. Capsaicin was applied at room
temperature for approximately 30 s before wash withexternal solution.
RR was diluted in external solution at the indicatedconcentrations and pre-incubated for at least 30 s before

Methods

co-application with heat (60˚C) or with 1 µM capsaicin.

Cells*Temperature given is temperature inside the pipette, temperature at cell was not

CHO cells stably expressing hTRPV1 were used.
Cell culture

measured and is likely to be 15 - 20˚C lower than pipette temperature indicated, i.e.
60˚C in pipette ~ 40 - 45ºC at cell.

Cells were cultured and harvested according to Nanion‘s standard cell culture protocol.
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