Ion Channels:

Application Note

TRPV3Cells:
HEK293Heat activation of TRPV3 on Nanion’s
Patchliner

Tools:Patchliner

Nanion Technologies GmbH, Munich

SummaryResults

The transient receptor potential cation channel, subfamily

Current responses of an individual cell to 200 ms voltage

V, member 3 (TRPV3), is a ligand-gated, non-selective

ramps (-100 mV to +100 mV) and activation by ­application­cation channel first described in 20021,2,3. It exhibits 43%

of increasing concentrations of 2-APB are shown in Figure 1.

sequence identity to TRPV11. Although TRPV3 has been

A concentration response curve (using amplitude at +90 mV)

detected immunologically in the CNS and suggested to berevealed an EC50 for 2-APB activation of 61.1 ± 7.6 µM (n =often co-localized with TRPV13, it is found more robustly in11), in good agreement with the literature4,5. Interestingly, as

keratinocytes in skin and, given its threshold for temperaturepreviously reported1,4, TRPV3 currents displayed sensitization toactivation of >34˚C1,2,3, it has been speculated that TRPV3

repeated application of 2-APB or heat (Figure 1).

may act in co-operation with sensory afferents to perceive
warmth and signal elevated temperature. TRPV3 canalso be activated by the ligand 2-Aminoethoxydiphenyl
borate (2-APB)4. The TRPV3 channel, along with other TRPV
channels, may play an important role in chronic pain and,
therefore, is receiving increasing attention as a potential
therapeutic candidate for the treatment of chronic pain.
Here we present data collected on a 4-channel P­ atchlinerwith temperature control showing the p­ otential use of thePatchliner to record TRPV3 currents activated by 2-APB or heat.

Figure 2 shows the activation of TRPV3 channels by increasing
temperature. Outwardly rectifying currents started to activate at38ºC and increased in amplitude as temperatures increased, up
to 54ºC, in good agreement with the literature1,2,3. To rule out theinvolvement of TRPV1 a control was performed using capsaicin.
Currents activated by 100 µM 2-APB were not activated by 1 µM
capsaicin (Figure 2B) as previously reported1.
A full concentration response curve (CRC) to RR at 0.1, 1, 3 and10 µM was performed (Figure 2C,D) using 100 µM 2-APB as theAs previously reported, TRPV3 currents sensitize to repeatedapplications of 2-APB4 or heat1, a phenomenon we alsoobserved. At low concentrations of 2-APB, the currents werewith prolonged exposure they often became dual rectifying

AB

3 µM 2-APB
30 µM 2-APB
100 µM 2-APB
300 µM 2-APB
500 µM 2-APB(data not shown). This is also in good agreement with theliterature4. In contrast, the temperature-activated responses
were always outwardly rectifying with little inward current.

1.00.8I/Imax

primarily outwardly rectifying but at higher concentrations andThe inward currents activated by 2-APB could be blocked byruthenium red (RR) as expected4.

0 nA-100 mV

2 nA

0.0

100 mVD

concentrations of 2-APB. B Concentration response
curve for 2-APB activation, EC50 = 61.1± 7.6 µM (n =11). C & D Repeated exposure of TRPV3 currents to2-APB (C) or temperature (D) caused sensitization ofcurrents (lines indicate 2-APB or heat exposure).

Amplitude (nA)Amplitude (nA)

12Figure 1: A Activation of TRPV3 by increasing

0.40.250 mV

C0.6

110100[2-APB (µM)]

2-APB

128400400
800
Time (s)1200

840200400 600
Time (s)800

Figure 2: A TRPV3 was activated by increasing

B

30°C
34°C
38°C
42°C
46°C
50°C
54°C

temperature. B TRPV3 was activated by 2-APB but

Control
100 µM 2-APB
1 µM capsaicin

not capsaicin. C The inward TRPV3 current wasblocked by RR in a concentration-dependent
manner but not the outward curreent (notshown). D CRC of the inward current (at -50 mV)
revealed an IC50 = 2.4 ± 0.4 µM (n = 4).

0 nA-100 mV

5 nA

C100 mV
50 mV-100 mV

0 nA

0 mV

0 nA

0.5 nA
20 mV

1 nA

D
1.0

50 mV

0.8

Control
100 nM RR
1µM RR
3 µM RR
10 µM RR

0.6I/Imax

A

0.40.20.0

1 nM 100 nM 10 µM[Ruthenium Red]

activator. As expected4, RR blocked the inward current with an

standard cell culture protocol. Cells were resuspended inIC50 of 2.4 ± 0.4 µM (n = 4). The outward currrent was not blocked

external recording solution and stored in the CellHotel ofby RR (not shown).

In summary, TRPV3 receptors stably expressed in HEK293 cells  can  be  reliably  activated  by  moderate (≥38˚C) to hot temperatures(up to 54˚C), or by 2-APB in a concentration-dependent manner.
The data shown here agrees well with published literature using
conventional patch clamp electrophysiology to study TRPV31-5.
Therefore, the Patchliner provides a viable, higher throughput
alternative to conventional patch clamp for the discovery ofactive TRPV3 lead compounds with a suitable drug profile. Whatis more, using the unique feature of the Patchliner which allows
rapid and transient activation of TRP channels by heating thesolution inside the pipette, compounds that block the ligand
response could be separated from those that block the heatactivated response.

References

the Patchliner before being dispensed into each well of theNPC-16 chip. Internal and external solution compositions areavailable upon request. Whole cell patch clamp recordings
were conducted according to Nanion’s standard procedure
for the Patchliner. Currents were elicited by 200 ms voltage
ramps from -100 mV to +100 mV, Vhold =  -20 mV. For heat

activation of channels, 100 µl of external solution was heatedin the pipette until desired temperature was reached (within 30s) and rapidly applied to the cell at a speed of 10 µl/s (N.B. acontrol application of 50 µM 2-APB at room temperature wasperformed at the start to ensure expression of TRPV3 in eachcell prior to heat application). 2-APB  was diluted in external
solution at the indicated concentrations and applied at roomtemperature for approximately 35 s before wash with externalsolution. RR was diluted in external solution at the indicatedconcentrations and pre-incubated for at least 30 s before coapplication with 100 µM 2-APB.

1. Peier, A.M., et al., 2002. Science. 296: 2046-2049.
2. Xu, H., et al., 2002. Nature. 418: 181-186.
3. Smith, G.D., et al., 2002. Nature. 418: 186-190.
4. Chung, M.K., et al., 2004. J. Neurosci. 24: 5177-5182.
5. Hu et al., 2009. PNAS. 106: 1626-1631.

Methods
Cells

PrecisION hTRPV3-HEK recombinant cell line (CYL3065) waskindly provided by EMD Millipore, USA.
Electrophysiology measurements
Cells were cultured and harvested according to Nanion’sNanion Technologies GmbH I Ganghoferstraße 70a, 80339 Munich, Germany+49 89 2190 95-0 I info@nanion.de I www.nanion.de